Methods, compositions, and kits relating to chitinases and chitinase-like molecules and inflammatory disease

ABSTRACT

The present invention includes compositions and methods for the treatment of inflammatory disease (e.g. asthma, COPD, inflammatory bowel disease, atopic dermatitis, atopy, allergy, allergic rhinitis, scleroderma, and the like), relating to inhibiting a chitinase-like molecule. The invention further includes methods to identify new compounds for the treatment of inflammatory disease, including, but not limited to, asthma, COPD and the like. This is because the present invention demonstrates, for the first time, that expression of IL-13, and of a chitinase-like molecule, mediates and/or is associated with inflammatory disease and that inhibiting the chitinase-like molecule treats and even prevents, the disease. Thus, the invention relates to the novel discovery that inhibiting a chitinase-like molecule treats and prevents an inflammatory disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.10/980,354, filed Nov. 3, 2004, which is a continuation of U.S. patentapplication Ser. No. 10/202,436, filed Jul. 23, 2002 (now U.S. Pat. No.7,214,373), which claims the benefit of U.S. Provisional Application No.60/307,432, filed Jul. 24, 2001, all of which applications areincorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was supported in part by funds obtained from the U.S.Government (National Institutes of Health Grant Numbers R01-HL-64242,R01-HL66571, and P01-HL-56389) and the U.S. Government may thereforehave certain rights in the invention.

BACKGROUND OF THE INVENTION

The prevalence of asthma has been steadily increasing for the past twodecades, with an estimated 17 million cases in the United States alone.Once believed to be primarily a dysfunction in the contractilemechanisms of airway smooth muscles, recent studies have indicated therole of the immune system and inflammation in asthma and other pulmonarydiseases.

Asthma is now characterized as a complex inflammatory disease attributedto the inappropriate stimulation of the immune system. In some cases,the inflammation is triggered by airborne antigens. In others, exogenoustriggers cannot be defined (intrinsic asthma). The immune cells andmediators implicated in asthmatic inflammation include IgE, mast cells,eosinophils, T cells, interleukin-4 (IL-4), IL-5, IL-9, IL-13 and othercytokines (Bradding et al., 1994, Am. J. Respir. Cell Mol. Biol.10:471-480; Bradding et al., 1997, Airway Wall Remodeling in Asthma, CRCPress, Boca Raton, Fla.; Nicolaides et al., 1997, Proc. Natl. Acad. Sci.USA 94:13175-13180; Wills-Karp, 1998, Science 282:2258-2260; Hamid etal., 1991, J. Clin. Invest. 87:1541-1546; Kotsimbos et al., 1996, Proc.Assoc. Am. Physicians 108:368-373). Of these immune cells and mediators,the role of T-helper type 2 (Th2) cells and cytokines is proving to beincreasingly important, as they are believed to be responsible forinitiation and maintenance of airway inflammation, as well as vital to Bcell regulation, eosinophil function, mucus responses, and stimulationof airway remodeling (Elias et al., 1999, J. Clin. Invest.104:1001-1006; Ray et al., 1999, J. Clin Invest. 104:985-993).

Immune-mediated inflammation is thought to lead to airway remodeling, orstructural modifications, in the asthmatic airway. The end result ofremodeling is believed to contribute to both the symptoms andphysiological dysregulation of asthma. Remodeling is often characterizedby airway thickening, mucus metaplasia, epithelial hypertrophy andairway fibrosis. Extensive fibrosis is widely considered to increasedisease severity, airway hyperresponsiveness (AHR) and contribute to thegeneration of incompletely reversible airway obstruction (Elias et al.,1999, J. Clin. Invest. 104:1001-1006). Therefore, the successful designof therapeutics for the treatment of asthma requires an understanding ofboth the mechanisms of inflammation and the processes of injury andwound healing in the respiratory system.

Two prominent cytokines, IL-4 and IL-13, are believed to play animportant role in the inflammation and airway remodeling of asthma andother pulmonary diseases. IL-4 and IL-13 are similar in that they areboth produced by the same subset of Th2 helper T cells, have overlappingeffector profiles, and share a receptor component and signalingpathways. However, the critical role of IL-13 over IL-4 in AHR,eosinophil recruitment, mucus overproduction, and other symptoms ofasthma has been conclusively demonstrated (Wills-Karp, 1998, Science282:2258-2260, Grunig et al. 1998, Science 282:2261-2263).Overexpression of IL-13 in the murine lung results in eosinophil,lymphocyte, and macrophage rich inflammation, mucus metaplasia, airwayfibrosis, and AHR after methacholine challenge (Zheng et al., 1999 J.Clin. Invest. 103:779-788). Further, polymorphisms in both the IL-13promoter and the coding region have been associated with the asthmaticphenotype (Heinzmann et al., 2000, Hum. Mol. Genet. 9:549-559). Theseresults suggest that abnormal IL-13 production is a critical componentof asthmatic inflammation and airway remodeling.

The role of IL-13 in inflammatory pulmonary diseases is not limited toasthma. Chronic obstructive pulmonary disease (COPD, clinically definedas chronic bronchitis, emphysema, and chronic obstructive lung disease)has long been thought of as a distinct disease from asthma. However, thesimilarities between the two diseases have been noted and have resultedin the formulation of the “Dutch Hypothesis”, that was first proposed in1961. The most recent revision of the Dutch Hypothesis proposes thatasthma and COPD, in some individuals, are not distinct processes, andthat common pathogenic mechanism underlie these disorders. Thehypothesis further states that a genetic predisposition to developatopy, asthma, AHR and/or increased levels of IgE predispose cigarettesmokers to develop COPD (Vestbo and Prescott, 1997, Lancet350:1431-1434). Further, overexpression of IL-13 in the murine lungcauses emphysema and COPD-like mucus metaplasia, IL-13 is overexpressedin biopsy and autopsy lung tissue from patients with COPD, andpolymorphisms of IL-13 have been described that correlate with thepresence of COPD. When these results are viewed in light of the DutchHypothesis, not only are asthma and COPD more closely related thanpreviously thought, but the central role of IL-13 dysregulation in thesepulmonary inflammatory disorders becomes more prominent.

The progress in illuminating the underlying mechanisms and causes ofasthma, COPD and related pulmonary inflammatory disorders is strikingconsidering the fact that what was once thought of as a malfunction ofbronchial muscle contraction can now be linked to specific cytokines andcell types. Despite this progress, asthma remains, along withtuberculosis and AIDS, the only chronic disease with an increasing deathrate. In addition, by 2020, COPD is expected to be the fourth leadingcause of death in the world.

To counter the increasing morbidity and mortality due to asthma, thearsenal of medications for the treatment of asthma is ever increasing.

Asthma medications fall into two general categories, controllers andrelievers. Controllers are for the prevention of asthma attacks beforesymptoms arise, and relievers are taken during the midst of an asthmaattack. Controllers include corticosteroids, widely considered the mostpotent and effective anti-inflammatory drugs available, cromolyn sodiumand nedocromil, milder anti-inflammatories often used in children, andlong-acting beta-2 agonists, which are bronchodilators. Relieversinclude short acting beta-2 agonists and anticholinergenics, which areoften used a supplements or alternatives to beta-2 agonists.

While corticosteroids and other therapeutics target theinflammatory-mediated symptoms of asthma, they often have broad-rangingimmunosuppressive properties, as well as other deleterious side effects.As the physiological and biological mechanisms of asthma are elucidated,development of specific and effective drugs should closely follow, andthe symptoms, morbidity, and mortality of asthma should drop, instead ofits current rise. However, despite increased understanding of theunderlying disease mechanism and despite the increasing incidence ofasthma, and morbidity and death therefrom, there are currently a limitednumber of effective and safe treatments for asthma, COPD and otherinflammatory diseases. In addition, there are no pharmacologic drugsthat alter the progression of COPD.

Thus, there is a long felt and acute need for specific, effectivetreatments for asthma, COPD, and other inflammatory diseases. Thepresent invention meets this need.

BRIEF SUMMARY OF THE INVENTION

A method of treating an inflammatory disease in a mammal wherein thedisease is associated with an increased level of a chitinase-likemolecule. The method comprises administering an effective amount of achitinase-like molecule inhibitor to the mammal, thereby treating theinflammatory disease in the mammal.

In one aspect, the mammal is a human.

In another aspect, the chitinase-like molecule is selected from thegroup consisting of a YM-1, a YM-2, an acidic mammalian chitinase(AMCase), an oviductal glycoprotein 1, a cartilage glycoprotein 1, achitotriosidase, a mucin 9, a cartilage glycoprotein-39, and achondrocyte protein 39.

In another aspect, the chitinase-like molecule inhibitor is selectedfrom the group consisting of a chemical compound, an antibody, aribozyme, a nucleic acid, and an antisense nucleic acid molecule.

In yet another aspect, the chemical compound is selected from the groupconsisting of allosamidin, glucoallosamidin A, glucoallosamidin B,methyl-N-demethylallosamidin, demethylallosamidin, didemthylallosamidin,stylogaunidine, a styloguanidine derivative, dipeptidecyclo-(L-Arg-D-Pro), dipeptide cyclo-(L-Arg-L-Pro), dipeptidecyclo-(D-Arg-D-Pro), dipeptide cyclo-(D-Arg-L-Pro), riboflavin, a flavinderivative, copper, zinc, and mercury.

In a further aspect, the antibody specifically binds with achitinase-like molecule selected from the group consisting of a YM-1, aYM-2, an acidic mammalian chitinase (AMCase), an oviductal glycoprotein1, a cartilage glycoprotein 1, a chitotriosidase, a mucin 9, a cartilageglycoprotein-39, and a chondrocyte protein 39.

In another aspect, the antisense nucleic acid molecule is an isolatednucleic acid complementary to an isolated nucleic acid encoding thechitinase-like molecule, or a fragment thereof.

In yet another aspect, the ribozyme is an isolated enzymatic nucleicacid, which specifically cleaves mRNA transcribed from a nucleic acidencoding the chitinase-like molecule.

In a further aspect, the inflammatory disease is selected from the groupconsisting of asthma, chronic obstructive pulmonary disease,interstitial lung disease, chronic obstructive lung disease, chronicbronchitis, eosinophilic bronchitis, eosinophilic pneumonia, pneumonia,inflammatory bowel disease, atopic dermatitis, atopy, allergy, allergicrhinitis, idiopathic pulmonary fibrosis, scleroderma, and emphysema.

The invention includes a method of preventing an inflammatory disease ina mammal wherein the disease is associated with an increased level of achitinase-like molecule. The method comprises administering an effectiveamount of a chitinase-like molecule inhibitor to the mammal, therebypreventing the inflammatory disease in the mammal.

In one aspect, the mammal is a human.

In another aspect, the chitinase-like molecule is selected from thegroup consisting of a YM-1, a YM-2, an acidic mammalian chitinase(AMCase), an oviductal glycoprotein 1, a cartilage glycoprotein 1, achitotriosidase, a mucin 9, a cartilage glycoprotein-39, and achondrocyte protein 39.

In another aspect, the chitinase-like molecule inhibitor is selectedfrom the group consisting of a chemical compound, an antibody, aribozyme, a nucleic acid, and an antisense nucleic acid molecule.

In a further aspect, the inflammatory disease is selected from the groupconsisting of asthma, chronic obstructive pulmonary disease,interstitial lung disease, chronic obstructive lung disease, chronicbronchitis, eosinophilic bronchitis, eosinophilic pneumonia, pneumonia,inflammatory bowel disease, atopic dermatitis, atopy, allergy, allergicrhinitis, idiopathic pulmonary fibrosis, scleroderma, and emphysema.

In yet another aspect, the chitinase-like molecule is a YM protein andfurther wherein the chitinase-like molecule inhibitor is selected fromthe group consisting of allosamidin, glucoallosamidin A,glucoallosamidin B, methyl-N-demethylallosamidin, demethylallosamidin,didemthylallosamidin, styloguanidine, a styloguanidine derivative,dipeptide cyclo-(L-Arg-D-Pro), dipeptide cyclo-(L-Arg-L-Pro), dipeptidecyclo-(D-Arg-D-Pro), dipeptide cyclo-(D-Arg-L-Pro), riboflavin, a flavinderivative, copper, zinc, and mercury.

In another aspect, the chitinase-like molecule is AMCase.

The invention includes a method of treating an inflammatory disease in amammal wherein the disease is associated with an increased level ofchitinase. The method comprising administering an effective amount of achitinase inhibitor to the mammal, thereby treating the inflammatorydisease in the mammal.

In one aspect, the mammal is a human.

In another aspect, the chitinase is acidic mammalian chitinase (AMCase)and the chitinase inhibitor is selected from the group consisting of achemical compound, an antibody, a ribozyme, a nucleic acid, a nucleicacid, and an antisense nucleic acid molecule.

In yet another aspect, the chemical compound is selected from the groupconsisting of allosamidin, glucoallosamidin A, glucoallosamidin B,methyl-N-demethylallosamidin, demethylallosamidin, didemthylallosamidin,stylogaunidine, a styloguanidine derivative, dipeptidecyclo-(L-Arg-D-Pro), dipeptide cyclo-(L-Arg-L-Pro), dipeptidecyclo-(D-Arg-D-Pro), dipeptide cyclo-(D-Arg-L-Pro), riboflavin, a flavinderivative, copper, zinc, and mercury.

In one aspect, the antibody specifically binds with AMCase.

In another aspect, the antisense nucleic acid molecule is an isolatednucleic acid complementary to an isolated nucleic acid encoding anAMCase, or a fragment thereof.

In yet another aspect, the inflammatory disease is selected from thegroup consisting of asthma, chronic obstructive pulmonary disease,interstitial lung disease, chronic obstructive lung disease, chronicbronchitis, eosinophilic bronchitis, eosinophilic pneumonia, pneumonia,inflammatory bowel disease, atopic dermatitis, atopy, allergy, allergicrhinitis, idiopathic pulmonary fibrosis, scleroderma, and emphysema.

The invention includes a method for treating an inflammatory disease ina mammal wherein the disease is associated with an increased level ofinterleukin-13. The method comprises administering an effective amountof a chitinase-like molecule inhibitor to the mammal, thereby treatingthe inflammatory disease in a mammal.

In one aspect, the mammal is a human.

In another aspect, the chitinase-like molecule inhibitor is selectedfrom the group consisting of a chemical compound, an antibody, aribozyme, a nucleic acid, and an antisense nucleic acid molecule.

The invention also includes a method for treating an inflammatorydisease in a mammal wherein the disease is associated with a Th2inflammatory response. The method comprises administering an effectiveamount of a chitinase-like molecule inhibitor to the mammal, therebytreating the inflammatory disease in a mammal.

The invention includes a method of identifying a compound useful fortreating an inflammatory disease in a mammal. The method comprisesadministering a compound to a mammal afflicted with an inflammatorydisease and comparing the level of a chitinase-like molecule in themammal with the level of the chitinase-like molecule in the mammal priorto administration of the compound, wherein a lower level of thechitinase-like molecule in the mammal after administration of thecompound compared with the level of the chitinase-like molecule in themammal prior to administration of the compound is an indication that thecompound is useful for treating an inflammatory disease in the mammal,thereby identifying a compound useful for treating an inflammatorydisease. In one aspect, the invention includes a compound identifiedusing this method.

In one aspect, the level of a chitinase-like molecule is selected fromthe group consisting of the level of chitinase-like molecule nucleicacid expression and the level of chitinase-like molecule enzymaticactivity.

In another aspect, the chitinase-like molecule is selected from thegroup consisting of a YM-1, a YM-2, an acidic mammalian chitinase(AMCase), an oviductal glycoprotein 1, a cartilage glycoprotein 1, achitotriosidase, a mucin 9, a cartilage glycoprotein-39, and achondrocyte protein 39.

In yet another aspect, the mammal is a mouse.

In a further aspect, the mouse is selected from the group consisting ofa transgenic mouse constitutively expressing interleukin 13 and atransgenic mouse inducibly expressing interleukin 13.

In another aspect, the chitinase-like molecule is AMCase. In yet anotheraspect, the invention includes a compound identified using this method.

The invention includes a method of identifying a compound useful fortreating an inflammatory disease. The method comprises contacting a cellwith a compound and comparing the level of a chitinase-like molecule inthe cell with the level of the chitinase-like molecule in an otherwiseidentical cell not contacted with the compound, wherein a lower level ofthe chitinase-like molecule in the cell contacted with the compoundcompared with the level of the chitinase-like molecule in the cell notcontacted with the compound is an indication that the compound is usefulfor treating an inflammatory disease, thereby identifying a compounduseful for treating an inflammatory disease.

The invention includes a kit for treating an inflammatory disease in amammal wherein the disease is associated with an increased level of achitinase-like molecule. The kit comprises an effective amount of achitinase-like molecule inhibitor, and further comprises an applicatorand an instructional material for the use thereof.

In one aspect, the chitinase-like molecule inhibitor is selected fromthe group consisting of a chemical compound, an antibody, a ribozyme, anantisense molecule, and a nucleic acid.

In another aspect, the chemical compound is selected from the groupconsisting of allosamidin, glucoallosamidin A, glucoallosamidin B,methyl-N-demethylallosamidin, demethylallosamidin, didemthylallosamidin,stylogaunidine, a styloguanidine derivative, dipeptidecyclo-(L-Arg-D-Pro), dipeptide cyclo-(L-Arg-L-Pro), dipeptidecyclo-(D-Arg-D-Pro), dipeptide cyclo-(D-Arg-L-Pro), riboflavin, a flavinderivative, copper, zinc, and mercury.

In yet another aspect, the chitinase-like molecule is AMCase.

The invention includes a kit for preventing an inflammatory disease in amammal wherein the disease is associated with an increased level of achitinase-like molecule. The kit comprises an effective amount of anchitinase-like molecule inhibitor, and further comprises an applicatorand an instructional material for the use thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

For the purpose of illustrating the invention, there are depicted in thedrawings certain embodiments of the invention. However, the invention isnot limited to the precise arrangements and instrumentalities of theembodiments depicted in the drawings.

FIG. 1 depicts the levels of IL-13 mRNA from autopsy lung tissues fromsmokers who died of COPD (S/C) and non-smokers without COPD (N), asdetermined by RT-PCR. The arrow indicates IL-13 transcripts.

FIG. 2, comprising FIG. 2A through FIG. 2D, is an image depictinghematoxylin and eosin (H&E) stained lung tissues from control normal andCC10-IL13 (constitutively expressing IL-13) mice. FIG. 2A (transgene(−), 100×) and FIG. 2B (transgene (+), 100×) illustrate histologictissue from the control and CC10-I1-13 mice respectively. FIG. 2Bdemonstrates the eosinophil, lymphocyte, and macrophage richinflammation in the parenchyma of the IL-13 overexpressing mice. FIG. 2C(transgene (−), 250×) and FIG. 2D (transgene (+), 250×) compare theairway epithelial cells of control normal mice and CC10-IL-13 transgenicmice respectively. FIG. 2D illustrates the epithelial hypertrophy in theairways of mice overexpressing IL-13.

FIG. 3, comprising FIG. 3A and FIG. 3B, is an image depicting periodicacid-Schiff with diastase (D-PAS) staining of airways from control andCC10-IL-13 mice. FIG. 3A depicts airways from transgene (−) mice, andFIG. 3B depicts mucus metaplasia and goblet cell hyperplasia intransgene (+) mice. Magnification is 100×.

FIG. 4, comprising FIG. 4A and FIG. 4B, depicts trichrome staining ofairways in CC10-IL-13 mice. FIG. 4A depicts collagen deposition intransgene (−) mice. FIG. 4B depicts enhanced collagen deposition intransgene (+) mice.

FIG. 5 is a schematic representation of the doxycycline (dox) inducibleconstructs used to generate the inducible CC10-rtTA-IL-13 transgenicmice.

FIG. 6, comprising FIG. 6A through 6E, depicts histologic andmorphometric analysis of lungs from CC10-rtTA-IL-13 inducible mice.Lungs were removed, fixed to pressure, and processed for microscopy toshow alveolar enlargement upon transgene induction after doxadministration. FIG. 6A depicts inducible transgene (+) mice before doxadministration. FIG. 6B depicts inducible transgene (+) mice one dayafter dox administration. FIG. 6C depicts inducible transgene (+) micealveoli 1 week after dox administration, and FIG. 6D shows considerablealveolar enlargement in inducible transgene (+) mice 1 month after doxadministration. FIG. 6E is a graph depicting the morphometric analysisof inducible transgene (+) mice. Four week old transgene (−) and (+)mice were randomized to normal or dox water and maintained on thesetreatments for one month. Lungs were removed, fixed to pressure, andchord length measurements were taken. Chord lengths were significantlylarger in transgene (+) mice given dox than in transgene (−) mice givendox or transgene (+) mice given normal water.

FIG. 7 depicts crystal deposition in transgenic mice constitutivelyexpressing IL-13. Crystals were multi-faceted and often needle-shaped.

FIG. 8 depicts a Coomassie blue stained SDS-PAGE gel comprisingpartially purified crystals from bronchioalveolar lavage (BAL) fluid ofCC10-IL-13 transgenic mice. The single protein bands had a molecularweight of approximately 45 kDa.

FIG. 9 depicts YM mRNA levels in the lungs of two month old transgene(+) and (−) CC10-IL-13 mice as determined by reverse transcriptasepolymerase chain reaction (RT-PCR).

FIG. 10, comprising FIGS. 10A and 10B, depicts YM mRNA levels intransgene (−) and (+) CC10-rtTA-IL-13 mice that were randomized tonormal or dox water starting at one month of age. In FIG. 10A, lungswere removed, after one month of randomization, total lung RNA wasisolated, and YM gene expression was determined using RT-PCR. FIG. 10Bdepicts YM mRNA after dox or normal water administration for theindicated time intervals (w=week, m=month). mRNA levels were normalizedusing β-actin as a control.

FIG. 11, comprising FIG. 11A through FIG. 11H, depicts in situhybridization of lung tissue from CC10-IL-13 and control mice to detectexpression of YM in the tissues. FIG. 11A and FIG. 11B depict lungtissue from transgene (+) mice probed with antisense and sense probes,respectively. FIG. 11C and FIG. 11D depict lung tissue from transgene(−) mice with antisense and sense probes, respectively. FIG. 11E andFIG. 11F depict lung tissue from four week old transgene (+) mice probedusing antisense and sense probes, respectively. FIG. 11G and FIG. 11Hdepict lung tissue from 10 week old transgene (+) mice probed withantisense and sense probes, respectively.

FIG. 12 is a graph depicting chitinase activity in BAL fluids obtainedfrom 2 month old transgene (−) and transgene (+) CC10-IL-13 animals.(p<0.05 versus transgene (−))

FIG. 13, comprising FIG. 13A and FIG. 13B, depicts acidic mammalianchitinase (AMCase) expression and chitinase activity in CC10-rtTA-IL-13mice, respectively. FIG. 13A depicts the levels of mRNA encoding AMCasesin lungs from transgene (−) and (+) mouse lungs that were placed on doxor normal water at one month of age and kept on this regimen for thenoted intervals. AMCase and β-actin expression were evaluated by RT-PCR.FIG. 13B is a graph depicting chitinase activity in inducible transgene(+) and (−) animals randomized to normal or dox water at one month ofage. BAL fluid chitinase activity was assessed at the noted intervalsafter dox or normal water administration.

FIG. 14, comprising FIG. 14A through 14D, depicts localization of AMCaseexpression in CC10-IL-13 mice using in situ hybridization. FIG. 14A andFIG. 14B depicts transgene (+) mouse lung tissue using antisense andsense probes, respectively. FIG. 14C and FIG. 14D depict transgene (−)mouse lung tissue using antisense and sense probes, respectively.

FIG. 15, comprising FIG. 15A through FIG. 15C, depicts YM and AMCasemRNA expression and chitinase activity in ovalbumin (OVA) sensitized andchallenged wild-type mice, as determined using RT-PCR and chitinaseactivity assays, respectively. FIG. 15A depicts YM mRNA expression atthe noted intervals after OVA aerosol challenge (d=days, h=hours). FIG.15B depicts AMCase mRNA expression at the noted intervals after OVAaerosol challenge. FIG. 15C depicts the significantly (*p<0.01) higherchitinase activity detected in bronchoalveolar lavage fluids (BAL) fromwild type mice twenty-four hours and later after OVA aerosol challenge.

FIG. 16, comprising FIG. 16A through 16G, depict the effects ofallosamidin (allos) on OVA sensitized and subsequently challenged wildtype animals. FIG. 16A is a graph depicting the effect of dailyallosamidin administration (1 mg/kg) on total BAL cell recovery.Asterisks indicate significant (p<0.01) reduction in total BAL cellrecovery after allosamidin administration. FIG. 16B is a graph depictingthe effect of daily allosamidin administration (1 mg/kg) on thepercentage of eosinophils recovered in BAL fluid. Asterisks indicatesignificant (p<0.01) reduction in the percentage of eosinophilsrecovered after allosamidin administration. FIG. 16C is a graphdepicting the effect of daily allosamidin administration (1 mg/kg) onthe total number of eosinophils recovered in BAL fluid. Asterisksindicate significant (p<0.01) reduction in total eosinophil recoveryafter allosamidin administration. FIG. 16D is a graph depicting theeffect of daily allosamidin administration (1 mg/kg) on the number oflymphocytes recovered in BAL fluid. Asterisks indicate significant(p<0.01) reduction in total lymphocyte recovery after allosamidinadministration. FIG. 16E is a graph depicting the dose dependent effectof allosamidin on total BAL cell recovery. Daily allosamidin doses weregiven starting on Day one, and animals were evaluated for total BAL cellrecovery on Days two and seven following OVA aerosol challenge.Asterisks indicate significant (p<0.01) reduction in total BAL cellrecovery after allosamidin administration. FIG. 16F is a graph depictingthe dose-dependent effect of allosamidin on the percentage eosinophilrecovery in BAL. Asterisks indicate significant (p<0.01) reduction inpercentage of eosinophils in BAL cell recovery after allosamidinadministration. FIG. 16G is a graph depicting the dose-dependent effectof allosamidin on the total eosinophil recovery in BAL. Asterisksindicate significant (p<0.01) reduction in total eosinophil BAL cellrecovery after allosamidin administration.

FIG. 17 depicts the effects of allosamidin administration (randomized to1 mg/kg allosamidin or vehicle control, daily doses intraperitoneally(i.p.) for 14 days) on lung size in five week old CC10-IL-13 transgene(+) and (−) mice. Lungs were removed, fixed to pressure, and assessedusing volume displacement methodology as described elsewhere herein.

FIG. 18, comprising FIGS. 18A and 18B, depicts the effects ofanti-AMCase antibodies on ovalbumin-induced BAL cell counts. FIG. 18A isa graph depicting the effect of anti-AMCase antibodies on totalovalbumin-induced BAL cell counts. Comparisons between unchallenged(unchall) and mice that were sensitized to ovalbumin and challenged onthree successive days with ovalbumin were made at a seven day timepoint. The mice were treated with 0.5 ml of anti-AMCase antibodies orcontrol serum (serum) intraperitoneally every other day starting the daybefore the first aerosol exposure. FIG. 18B is a graph depicting theeffect of anti-AMCase antibodies on the percentage of eosinophils in BALfluids from our sensitized and challenged mice. Comparisons betweenunchallenged (unchall) and mice that were sensitized to ovalbumin andchallenged on three successive days with ovalbumin were made at a sevenday time point. The mice were treated with 0.5 ml of anti-AMCaseantibodies or control serum (serum) intraperitoneally every other daystarting the day before the first aerosol exposure.

FIG. 19 comprises FIGS. 19A through 19F. FIG. 19A through 19D are imagesdepicting detectable AMCase mRNA using in situ hybridization in autopsylung samples from a patient with asthma using an AMCase antisense probe(FIG. 19A). AMCase mRNA was not detected in histologically normalcontrol lung tissue obtained at autopsy from patients without lungdisease using the antisense probe (FIG. 19B). In situ hybridizationusing a sense probe did not detect AMCase in either fatal asthma tissue(FIG. 19C) or control tissue (FIG. 19D). Epithelial cell (thick arrow)and macrophage (thin arrow) staining in fatal asthma was detected (FIG.19A). FIGS. 19E and 19F are images depicting detectable AMCase mRNAusing in situ hybridization in alveolar macrophages present in autopsylung samples from patients with asthma using an AMCase antisense probe(FIG. 19E). AMCase mRNA was not detected using a sense probe (FIG. 19F).Further, AMCase mRNA was not detected in control lung tissue obtainedfrom patients without lung disease using the antisense or sense probes.

DETAILED DESCRIPTION OF THE INVENTION

The invention includes a method of treating an inflammatory disease in amammal where the disease is associated with, or mediated by, expressionof a chitinase-like molecule. The method comprises administering achitinase-like molecule inhibitor to the mammal. As the data disclosedelsewhere herein demonstrate, increased level of a chitinase-likemolecule, is associated with, and/or mediates an inflammatory diseaseincluding, but not limited to, asthma, chronic obstructive pulmonarydisease, interstitial lung disease, chronic obstructive lung disease,chronic bronchitis, eosinophilic bronchitis, eosinophilic pneumonia,pneumonia, inflammatory bowel disease, atopic dermatitis, atopy,allergy, allergic rhinitis, idiopathic pulmonary fibrosis, scleroderma,emphysema, and the like.

The data disclosed herein demonstrate that increased expression,presence and/or activity of a chitinase-like molecule is associated withand/or mediates various inflammatory disease-associated etiologiesincluding, but not limited to, tissue inflammation, increased lungvolume, increased eosinophils in bronchoalveolar lavage (BAL) fluid,increased lymphocytes in BAL fluid and tissues, increased total cells inBAL fluid, increased alveolus size, increased airway resistance,increased mucus metaplasia, increased mucin expression, increasedparenchymal fibrosis, increased airway remodeling, increasedsubepithelial fibrosis, increased collagen deposition in airway tissue,epithelial hypertrophy in the lung tissue, focal organization ofcrystalline material into Masson body-like fibrotic foci, and the like.

The data disclosed herein demonstrate, surprisingly, that even thoughsome chitinase-like molecules, for example, YM, do not have detectableclassical chitinase activity, administering a chitinase-like moleculeinhibitor, such as, but not limited to, allosamidin, provides atherapeutic benefit and treats the disease. Further, the data disclosedherein demonstrate, for the first time, that administration an inhibitorof a chitinase-like molecule, e.g. an antibody to AMCase, provides atherapeutic effect and treats the disease. Indeed, the data demonstratethat administration of a chitinase-like molecule inhibitor before onsetof the disease state serves to prevent the disease. Accordingly, thepresent invention provides a novel method whereby administration of achitinase-like molecule inhibitor in a mammal afflicted with aninflammatory disease treats and/or prevents the disease when the diseaseis mediated by, or associated with, a chitinase-like molecule, eventhough the chitinase-like molecule may or may not have detectablechitinase activity.

DEFINITIONS

As used herein, each of the following terms has the meaning associatedwith it in this section.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e. to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

By the term “applicator” as the term is used herein, is meant any deviceincluding, but not limited to, a hypodermic syringe, a pipette, anintravenous infusion, topical cream and the like, for administering thechitinase-like molecule inhibitor chemical compound, an antibody,nucleic acid, protein, and/or composition of the invention to a mammal.

“Chitinase,” as used herein, refers to a family of polypeptidescomprising microbial and mammalian chitinases. A chitinase of thepresent invention demonstrates detectable chitinase activity, in that itspecifically cleaves chitin in an endochitinase manner.

“Chitinase-like molecule,” as the term is used herein, encompasses afamily of polypeptides comprising proteins that are defined by a certaindegree of homology to known chitinases, but may not demonstratedetectable chitinase activity. Chitinase-like molecules include, but arenot limited to acidic mammalian chitinase (also referred to aseosinophil chemotactic cytokine and exemplified by GenBank Acc. No.AF290003 and No. AF29004), YM1 (also known as chitinase 3-like 3, ECF-Lprecursor, as exemplified by GenBank Acc. No. M94584), YM2 asexemplified by GenBank Acc. No. AF461142, oviductal glycoprotein 1 asexemplified by GenBank Acc. No. XM_(—)131100, cartilage glycoprotein 1(also referred to as BRP-39, chitinase 3-like 1, GP-1-39, YKL-40 andexemplified by GenBank Acc. No. X93035), chitotriosidase as exemplifiedby GenBank Acc. No. NM_(—)003465, oviductal glycoprotein 1 (alsoreferred to as mucin 9, oviductin and as exemplified by GenBank Acc. No.NM_(—)002557), cartilage glycoprotein-39 (also known as chitinase 3-like1, GP-39, YKL-40, as exemplified by GenBank Acc. No. NM_(—)001276), andchondrocyte protein 39 (also known as chitinase 3-like 2, YKL-39, asexemplified by GenBank Acc. No. NM_(—)004000). Thus, the skilled artisanwould appreciate, once armed with the teachings provided herein, thatthe present invention encompasses chitinase-like molecules that possessdetectable chitinase activity as well as those similar to theafore-mentioned molecules in that the potential chitinase-like moleculesshares substantial sequence homology to the family of proteins. Theinvention is not limited to these particular chitinase-like molecules;rather, the invention includes other chitinase-like molecules that sharesubstantial homology with them and/or which possess detectable chitinaseactivity, and encompasses such molecules known in the art as well asthose discovered in the future.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA corresponding to thatgene produces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and thenon-coding strand, used as the template for transcription of a gene orcDNA, can be referred to as encoding the protein or other product ofthat gene or cDNA.

As used herein, the term “fragment” as applied to a nucleic acid, mayordinarily be at least about 20 nucleotides in length, typically, atleast about 50 nucleotides, more typically, from about 50 to about 200nucleotides, preferably, at least about 200 to about 300 nucleotides,even more preferably, at least about 300 nucleotides to about 400nucleotides, yet even more preferably, at least about 400 to about 500,even more preferably, at least about 500 nucleotides to about 600nucleotides, yet even more preferably, at least about 600 to about 700,even more preferably, at least about 700 nucleotides to about 800nucleotides, yet even more preferably, at least about 800 to about 900,even more preferably, at least about 900 nucleotides to about 1000nucleotides, yet even more preferably, at least about 1000 to about1100, even more preferably, at least about 1100 nucleotides to about1200 nucleotides, yet even more preferably, at least about 1200 to about1300, even more preferably, at least about 1300 nucleotides to about1400 nucleotides, yet even more preferably, at least about 1400 to about1500, at least about 1500 to about 1550, even more preferably, at leastabout 1550 nucleotides to about 1600 nucleotides, yet even morepreferably, at least about 1600 to about 1620 and most preferably, thenucleic acid fragment will be greater than about 1625 nucleotides inlength.

“Homologous” as used herein, refers to the subunit sequence similaritybetween two polymeric molecules, e.g. between two nucleic acidmolecules, e.g., two DNA molecules or two RNA molecules, or between twopolypeptide molecules. When a subunit position in both of the twomolecules is occupied by the same monomeric subunit, e.g., if a positionin each of two DNA molecules is occupied by adenine, then they arehomologous at that position. The homology between two sequences is adirect function of the number of matching or homologous positions, e.g.if half (e.g., five positions in a polymer ten subunits in length) ofthe positions in two compound sequences are homologous then the twosequences are 50% homologous, if 90% of the positions, e.g. 9 of 10, arematched or homologous, the two sequences share 90% homology. By way ofexample, the DNA sequences 3′-ATTGCC-5′ and 3′-TATGGC-5′ share 75%homology.

By “chitinase-like molecule inhibitor” is meant a compound thatdetectably inhibits the level of a chitinase-like molecule in a cell ortissue when compared to the level of the chitinase-like molecule in anotherwise identical cell or tissue in the absence of the compound. Thelevel of the chitinase-like molecule includes, but is not limited to,the level of expression of a nucleic acid encoding the molecule, thelevel of chitinase-like molecule detectable, and/or the level ofchitinase activity. Chitinase-like molecule inhibitors include, but arenot limited to, a chemical compound (e.g., allosamidin, glucoallosamidinA, glucoallosamidin B, methyl-N-demethylallosamidin,demethylallosamidin, didemthylallosamidin, stylogaunidine, astyloguanidine derivative, dipeptide cyclo-(L-Arg-D-Pro), dipeptidecyclo-(L-Arg-L-Pro), dipeptide cyclo-(D-Arg-D-Pro), dipeptidecyclo-(D-Arg-L-Pro), riboflavin, a flavin derivative), copper, zinc,mercury, an antibody, a ribozyme, an antisense molecule, and a nucleicacid.

An “AMCase inhibitor,” as the term is used herein, includes achitinase-like molecule inhibitor, as defined previously, that inhibitsAMCase. Such inhibitor includes, but it not limited to, a chemicalcompound, as well as a ribozyme, antisense molecule, an antibody, andthe like, that inhibits the level of AMCase expression and/or activityin a cell or tissue compared with the level of AMCase expression and/oractivity in the cell or tissue in the absence of the inhibitor, or in anotherwise identical cell or tissue, in the absence of the inhibitor. Theinhibitor includes, but is not limited to, a chemical compound, aribozyme, an antisense nucleic acid molecule, an antibody, and the like.

As used herein, the terms “gene” and “recombinant gene” refer to nucleicacid molecules comprising an open reading frame encoding a polypeptideof the invention. Such natural allelic variations can typically resultin 1-5% variance in the nucleotide sequence of a given gene. Alternativealleles can be identified by sequencing the gene of interest in a numberof different individuals. This can be readily carried out by usinghybridization probes to identify the same genetic locus in a variety ofindividuals. Any and all such nucleotide variations and resulting aminoacid polymorphisms or variations that are the result of natural allelicvariation and that do not alter the functional activity are intended tobe within the scope of the invention.

As used herein, an “instructional material” includes a publication, arecording, a diagram, or any other medium of expression, which can beused to communicate the usefulness of the nucleic acid, peptide, and/orcomposition of the invention in the kit for effecting alleviation of thevarious diseases or disorders recited herein. Optionally, oralternately, the instructional material may describe one or more methodsof alleviation the diseases or disorders in a cell or a tissue of amammal. The instructional material of the kit of the invention may, forexample, be affixed to a container, which contains the nucleic acid,peptide, chemical compound and/or composition of the invention or beshipped together with a container, which contains the nucleic acid,peptide, chemical composition, and/or composition. Alternatively, theinstructional material may be shipped separately from the container withthe intention that the instructional material and the compound be usedcooperatively by the recipient.

An “inflammatory disease” is used herein to refer to a state in whichthere is a response to tissue damage, cell injury, an antigen, and/or aninfectious disease. In some cases, causation will not be able to beestablished. The symptoms of inflammation may include, but are notlimited to cell infiltration and tissue swelling. Disease statescontemplated under the definition of inflammatory disease includeasthma, chronic obstructive pulmonary disease, interstitial lungdisease, chronic obstructive lung disease, chronic bronchitis,eosinophilic bronchitis, eosinophilic pneumonia, pneumonia, inflammatorybowel disease, atopy dermatitis, atopy, allergy, allergic rhinitis,idiopathic pulmonary fibrosis, scleroderma, and emphysema.

An “isolated nucleic acid” refers to a nucleic acid segment or fragmentwhich has been separated from sequences which flank it in a naturallyoccurring state, e.g. a DNA fragment which has been removed from thesequences which are normally adjacent to the fragment, e.g. thesequences adjacent to the fragment in a genome in which it naturallyoccurs. The term also applies to nucleic acids, which have beensubstantially purified from other components, which naturally accompanythe nucleic acid, e.g., RNA or DNA or proteins, which naturallyaccompany it in the cell. The term therefore includes, for example, arecombinant DNA which is incorporated into a vector, into anautonomously replicating plasmid or virus, or into the genomic DNA of aprokaryote or eukaryote, or which exists as a separate molecule (e.g.,as a cDNA or a genomic or cDNA fragment produced by PCR or restrictionenzyme digestion) independent of other sequences. It also includes arecombinant DNA, which is part of a hybrid gene encoding additionalpolypeptide sequence.

By describing two polynucleotides as “operably linked” is meant that asingle-stranded or double-stranded nucleic acid moiety comprises the twopolynucleotides arranged within the nucleic acid moiety in such a mannerthat at least one of the two polynucleotides is able to exert aphysiological effect by which it is characterized, upon the other. Byway of example, a promoter operably linked to the coding region of agene is able to promote transcription of the coding region.

Preferably, when the nucleic acid encoding the desired protein furthercomprises a promoter/regulatory sequence, the promoter/regulatorysequence is positioned at the 5′ end of the desired protein codingsequence such that it drives expression of the desired protein in acell. Together, the nucleic acid encoding the desired protein and itspromoter/regulatory sequence comprise a “transgene.”

“Constitutive” expression is a state in which a gene product is producedin a living cell under most or all physiological conditions of the cell.

“Inducible” expression is a state in which a gene product is produced ina living cell in response to the presence of a signal in the cell.

A “recombinant polypeptide” is one, which is produced upon expression ofa recombinant polynucleotide.

“Polypeptide” refers to a polymer composed of amino acid residues,related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof linked via peptide bonds,related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof. Synthetic polypeptides can besynthesized, for example, using an automated polypeptide synthesizer.

The term “protein” typically refers to large polypeptides.

The term “peptide” typically refers to short polypeptides.

As used herein, the term “transgenic mammal” means a mammal, the germcells of which, comprise an exogenous nucleic acid.

As used herein, to “treat” means reducing the frequency with whichsymptoms of the inflammatory disease, are experienced by a patient, oraltering the natural history and/or progression of the disease in apatient.

As used herein, the term “antisense oligonucleotide” means a nucleicacid polymer, at least a portion of which is complementary to a nucleicacid which is present in a normal cell or in an affected cell. Mostpreferably, the antisense oligonucleotides comprise between aboutfifteen and about fifty nucleotides. The antisense oligonucleotides ofthe invention include, but are not limited to, phosphorothioateoligonucleotides and other modifications of oligonucleotides.

The term “antibody,” as used herein, refers to an immunoglobulinmolecule which is able to specifically bind to a specific epitope on anantigen. Antibodies can be intact immunoglobulins derived from naturalsources or from recombinant sources and can be immunoreactive portionsof intact immunoglobulins. Antibodies are typically tetramers ofimmunoglobulin molecules. The antibodies in the present invention mayexist in a variety of forms including, for example, polyclonalantibodies, monoclonal antibodies, Fv, Fab and F(ab)₂, as well as singlechain antibodies and humanized antibodies (Harlow et al., 1999, UsingAntibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,NY; Harlow et al., 1989, Antibodies: A Laboratory Manual, Cold SpringHarbor, N.Y.; Houston et al., 1988, Proc. Natl. Acad. Sci. USA85:5879-5883; Bird et al., 1988, Science 242:423-426).

By the term “synthetic antibody” as used herein, is meant an antibodywhich is generated using recombinant DNA technology, such as, forexample, an antibody expressed by a bacteriophage as described herein.The term should also be construed to mean an antibody which has beengenerated by the synthesis of a DNA molecule encoding the antibody andwhich DNA molecule expresses an antibody protein, or an amino acidsequence specifying the antibody, wherein the DNA or amino acid sequencehas been obtained using synthetic DNA or amino acid sequence technologywhich is available and well known in the art.

A “portion” of a polynucleotide means at least at least about fifteen toabout fifty sequential nucleotide residues of the polynucleotide. It isunderstood that a portion of a polynucleotide may include everynucleotide residue of the polynucleotide.

By the term “specifically binds,” as used herein, is meant an antibodywhich recognizes and binds a chitinase-like molecule, but does notsubstantially recognize or bind other molecules in a sample.

A “prophylactic” treatment is a treatment administered to a subject whodoes not exhibit signs of a disease or exhibits only early signs of thedisease for the purpose of decreasing the risk of developing pathologyassociated with the disease.

“Preventing” a disease, as the term is used herein, means that the onsetof the disease is delayed, and/or that the symptoms of the disease willbe decreased in intensity and/or frequency, when a chitinase-likemolecule is administered compared with the onset and/or symptoms in theabsence of the inhibitor.

A “therapeutic” treatment is a treatment administered to a subject whoexhibits signs of pathology for the purpose of diminishing oreliminating those signs.

I. Methods

A. Methods of Treating an Inflammatory Disease

The present invention includes a method of treating an inflammatorydisease wherein the disease is associated with an increased level of achitinase-like molecule. Contemplated in the present invention aremethods of treating an inflammatory disease in a mammal, preferably ahuman, using a chitinase-like molecule inhibitor. This is because, aswould be appreciated by one skilled in the art when provided with thedisclosure herein, inhibiting the expression and/or activity of achitinase-like molecule serves as a treatment for inflammatory diseases,including diseases mediated by IL-13. That is, the data disclosed hereindemonstrate that administration of a chitinase-like molecule inhibitorin a model of inflammatory disease associated with, or mediated by,expression of IL-13, treats the disease after it has become established.Further, the present invention relates to the discovery thatchitinase-like molecules and chitinase-like molecule mRNA are present inincreased levels in inflammatory disease tissue compared with the levelof a chitinase-like molecule in normal tissue. Thus, the presentinvention relates to treating of such diseases using a chitinase-likemolecule inhibitors, including, but not limited to, a chitinase-likemolecule inhibitor (e.g. allosamidin).

Surprisingly, a chitinase-like molecule inhibitor can be administered totreat the disease even when there is no detectable chitinase activity.Thus, the skilled artisan would appreciate, based upon the disclosureprovided herein, that the present invention is not limited to treatmentof a disease where detectable chitinase activity is present; instead,the present invention encompasses treatment of a disease associated withor mediated by expression of a chitinase-like molecule even when thereis no detectable chitinase activity.

It would be understood by one skilled in the art, based upon thedisclosure provided herein, that inhibition of a chitinase-like moleculeencompasses inhibition of a chitinase-like molecule expression, such asthat mediated by, among other things, a ribozyme and/or antisensemolecule that inhibits expression of a nucleic acid encoding achitinase-like molecule. Additionally, the skilled artisan wouldappreciate, once armed with the teachings of the present invention, thatinhibition of a chitinase-like molecule includes inhibition of achitinase-like molecule activity in a cell. Such inhibition of achitinase-like molecule activity can be effected using inhibitors ofchitinase enzymatic activity, including, inter alia, allosamidin,1,10-phenanthroline, glucoallosamidin A, glucoallosamidin B,methyl-N-demethylallosamidin, demethylallosamidin, didemthylallosamidin,stylogaunidine, a styloguanidine derivative, dipeptidecyclo-(L-Arg-D-Pro), dipeptide cyclo-(L-Arg-L-Pro), dipeptidecyclo-(D-Arg-D-Pro), dipeptide cyclo-(D-Arg-L-Pro), riboflavin, a flavinderivative, copper, zinc, mercury and the like. Further, inhibitors ofchitinase-like molecule activity include an antibody that specificallybinds with a chitinase-like molecule thereby preventing the enzyme fromfunctioning. Thus, a chitinase-like molecule inhibitor includes, but isnot limited to, inhibiting transcription, translation, or both, of anucleic acid encoding a chitinase-like molecule; and it also includesinhibiting any activity of the peptide as well, including, but notlimited to, the ability to cleave chitin.

The present invention includes a method of treating or preventing aninflammatory disease in a mammal. The method comprises administering achitinase-like molecule inhibitor to a mammal in need of such treatment.This is because, as would be appreciated by one skilled in the art armedwith the teachings of the present invention, inhibiting a chitinase-likemolecule is useful for treating or preventing an inflammatory disease.Inhibition of a chitinase-like molecule prevents, in turn, the pathologyassociated with an inflammatory disease, as amply demonstrated by thedata disclosed herein.

More specifically, the invention relates to inhibiting a chitinase-likemolecule using various inhibitors. That is, one skilled in the art wouldunderstand, based upon the disclosure provided herein, that compoundsthat inhibit the expression, activity, and/or function of achitinase-like molecule encompass, but are not limited to, an antibody,an antisense nucleic acid, a ribozyme, a small molecule, apeptidomimetic and a chemical compound, either known or to be developed,which inhibits a chitinase-like molecule, and thereby an inflammatorydisease.

The invention encompasses inhibition of a chitinase-like molecule wherethe molecule does or does not demonstrate detectable chitinase activityin vitro or in vivo. That is, without wishing to be bound by anyparticular theory, whether the chitinase-like molecule demonstratesdetectable chitinase activity, either in a cell or tissue or in acell-free system, is not relevant. More specifically, as demonstrated bythe data disclosed herein, a chitinase-like molecule, such as Ym, whichdoes not demonstrate detectable chitinase activity in vitro or in vivo,is expressed at an increased level in disease cells and tissues comparedto a cell or tissue that does not demonstrate inflammatory diseasepathology, and inhibiting Ym using, inter alia, allosamidin, treatsand/or prevents the disease. Additionally, the data disclosed hereinfurther demonstrate that increased level of a chitinase, AMCase, in acell or tissue is associated with, or mediates, an inflammatory disease.Moreover, inhibition of AMCase treats the disease. Such therapeuticeffect may be related to inhibition of undetectable chitinase activity,or it may be that the therapeutic effect does not relate to anychitinase or chitinase-like activity of the chitinase-like molecule, andthe skilled artisan, based upon the disclosure provided herein, wouldappreciate that the therapeutic effect can be, but need not be,correlated with any chitinase activity by the molecule.

One skilled in the art would appreciate, based on the disclosureprovided herein, that an inhibitor of the invention includes moleculesand compounds that prevent or inhibit the expression, activity orfunction of a chitinase-like molecule in a mammal. That is, theinvention contemplates that an antisense and/or antisense molecule thatinhibits, decreases, and/or abolishes expression of a chitinase-likemolecule such that the chitinase-like molecule is not detectable in thecell or tissue is an inhibitor of the invention. For instance, acompound that degrades a chitinase-like molecule can decrease itsfunction, and can be an inhibitor as contemplated in the presentinvention.

Inhibition of a chitinase-like molecule can be assessed using a widevariety of methods, including those disclosed herein, as well as methodswell-known in the art or to be developed in the future. That is, theroutineer would appreciate, based upon the disclosure provided herein,that inhibition of chitinase-like molecule expression can be readilyassessed using methods that assess the level of a nucleic acid encodinga chitinase-like molecule (e.g., mRNA) and/or the level of achitinase-like molecule present in a cell or fluid. Moreover, theroutineer would understand that inhibition of a chitinase-like moleculecan be assessed by determining the inhibition of chitinase enzymaticactivity in a cell or bodily fluid as exemplified elsewhere hereinand/or using methods well-known in the art or to be developed in thefuture.

One skilled in the art, based upon the disclosure provided herein, wouldunderstand that the invention encompasses treatment of a variety ofinflammatory diseases, including, but not limited to, asthma, chronicobstructive pulmonary disease, interstitial lung disease, chronicobstructive lung disease, chronic bronchitis, eosinophilic bronchitis,eosinophilic pneumonia, pneumonia, inflammatory bowel disease, atopicdermatitis, atopy, allergy, allergic rhinitis, idiopathic pulmonaryfibrosis, scleroderma, and emphysema, and the like. As disclosed herein,these diseases involve and/or are mediated by, increased chitinase-likemolecules in tissues where increased chitinase-like molecules includes,and is not limited to, increased chitinase-like molecule expression,increased chitinase-like molecule activity, or both.

Further, the skilled artisan would further appreciate, based upon theteachings provided herein, that the diseases encompass any diseasecomprising increased chitinase-like molecule in a tissue including,among others, a disease mediated by increased IL-13 and/or increasedIL-4 production. This is because, as more fully set forth elsewhereherein, the data disclosed herein demonstrate that increased IL-13and/or increased IL-4 mediates an increase in chitinase-like moleculeswhich, in turns, mediates and/or is associated with a variety of changesassociated with inflammatory disease including, but not limited to,tissue inflammation, increased lung volume, increased eosinophils inbronchioalveolar lavage (BAL) fluid, increased lymphocytes in BAL fluid,increased total cells in BAL fluid, increased alveolus size, increaseddeposition of crystals comprising chitinase-like molecules in lungtissue, increased airway resistance, increased mucus metaplasia,increased mucin expression, increased parenchymal fibrosis, increasedairway remodeling, increased subepithelial fibrosis, increased collagendeposition in airway tissue, epithelial hypertrophy in the lung tissue,focal organization of crystalline material into Masson body-likefibrotic foci, and the like.

Therefore, the data disclosed herein demonstrate that inhibition of achitinase-like molecule in a mammal afflicted with an inflammatorydisease, wherein the disease is mediated or associated with increasedexpression of IL-13 and/or IL-4, will treat the disease by mediating adecrease in the level of a chitinase-like molecule which, in turn,treats the disease. For instance, such data include, but are not limitedto, the inhibition of various tissue pathology by administering achitinase-like molecule inhibitor (e.g., allosamidin) to a mammal whereincreased expression of IL-13 mediates increased chitinase-like moleculeactivity and increased chitinase-like molecule expression.

The present invention further comprises a method for treating aninflammatory disease mediated by and/or associated with a Th2inflammatory response in a mammal. The skilled artisan, when armed withthe present disclosure and the teachings provided herein, wouldunderstand that an inflammatory disease mediated by and/or associatedwith a Th2 inflammatory response encompasses a variety of inflammatorydiseases, including, but not limited to, asthma, chronic obstructivepulmonary disease, interstitial lung disease, chronic obstructive lungdisease, chronic bronchitis, eosinophilic bronchitis, eosinophilicpneumonia, pneumonia, inflammatory bowel disease, atopic dermatitis,atopy, allergy, allergic rhinitis, idiopathic pulmonary fibrosis,scleroderma, and emphysema, and the like. As disclosed herein, thesediseases are mediated by a Th2 inflammatory response in an mammal, andresult in, among other things, increased IL-13 production and/orexpression, increased chitinase-like molecule activity and/orexpression, and the like. Increased chitinase-like molecules include,but is not limited to, increased chitinase-like molecule expression,increased chitinase-like molecule activity, or both.

Further, the skilled artisan would appreciate, based upon the teachingsprovided herein, that the diseases encompass any disease comprisingincreased chitinase-like molecule in a tissue including, among others, adisease mediated by increased Th2 inflammatory response. This isbecause, as more fully set forth elsewhere herein, the data disclosedherein demonstrate that increased Th2 inflammatory responses result in,inter alia, increased IL-13 and/or increased IL-4 activity and/orexpression, an increase in chitinase-like molecules which, in turns,mediates and/or is associated with a variety of changes associated withinflammatory disease including, but not limited to, increased totalcells in BAL fluid, increased alveolus size, increased deposition ofcrystals comprising chitinase-like molecules in lung tissue, increasedairway resistance, increased mucus metaplasia, increased mucinexpression, increased parenchymal fibrosis, increased airway remodeling,increased subepithelial fibrosis, increased eosinophils inbronchioalveolar lavage (BAL) fluid, increased lymphocytes in BAL fluid,and the like.

Therefore, the data disclosed herein demonstrate that inhibition of achitinase-like molecule in a mammal afflicted with an inflammatorydisease, wherein the disease is mediated by and/or associated with anincreased Th2 inflammatory response, will treat the disease by mediatinga decrease in the level of a chitinase-like molecule which, in turn,treats the disease. For instance, such data include, but are not limitedto, the inhibition of various tissue pathology by administering achitinase-like molecule inhibitor (e.g., allosamidin) to a mammal wherea Th2 inflammatory response mediates increased chitinase-like moleculeactivity and increased chitinase-like molecule expression.

The skilled artisan will further appreciate that a chitinase-likemolecule is a molecule that exhibits a substantial degree of homology toknown chitinases, such that it has been or can be classified as achitinase family molecule based upon, inter alia, its amino acidsequence. Further, the skilled artisan would understand, based upon thedisclosure provided herein, that while a chitinase-like molecule canexhibit homology to a known chitinase, a chitinase-molecule need notdemonstrate detectable chitinase activity, in that they may notdetectably cleave chitin an in assay known in the art. Such chitinaselike molecules include, but are not limited to, acidic mammalianchitinase (eosinophil chemotactic cytokine), YM1 (chitinase 3-like 3,ECF-L precursor), YM2, oviductal glycoprotein 1, cartilage glycoprotein1 (BRP-39, chitinase 3-like 1, GP-39, YKL-40), chitotriosidase,oviductal glycoprotein 1 (mucin 9, oviductin), cartilage glycoprotein-39(chitinase 3-like 1, GP-39, YICL-40), and chondrocyte protein 39(chitinase 3-like 2, YKL-39).

A chitinase-like molecule inhibitor can include, but should not beconstrued as being limited to a chemical compound, a protein, apeptidomemetic, an antibody, a ribozyme, and an antisense nucleic acidmolecule.

One of skill in the art would readily appreciate, based on thedisclosure provided herein, that a chitinase-like molecule inhibitorencompasses a chemical compound that inhibits the activity of achitinase-like molecule. Chitinase-like molecule inhibitors are wellknown in the art, and some of the key critical elements of one class ofchitinase-like molecule inhibitors have been defined (Spindler andSpindler-Barth, 1999, Chitin and Chitinases, Birkhauser Verlag Basel,Switzerland). Additionally, a chitinase-like molecule inhibitorencompasses a chemically modified compound, and derivatives, as is wellknown to one of skill in the chemical arts.

The skilled artisan would appreciate that a chitinase-like moleculeinhibitor encompasses an already known chitinase-like molecule inhibitorsuch as, but not limited to, allosamidin (Allosamidine, CarbohydrateChemistry Industrial Research Limited, Lower Hutt, New Zealand, and EliLilly and Co., Greenfield, Ind.) and its derivatives (see, e.g., U.S.Pat. No. 5,413,991), glucoallosamidin A, glucoallosamidin B,methyl-N-demethylallosamidin (Nishimoto et al., 1991, J. Antibiotics44:716-722) demethylallosamidin (U.S. Pat. No. 5,070,191), anddidemthylallosamidin (Zhou et al., 1993, J. Antibiotics 46:1582-1588).Further contemplated chitinase-like molecule inhibitors includestylogaunidine and its derivatives (Kato et al., 1995, Tetrahedron.Lett. 36:2133-2136), dipeptide cyclo-(L-Arg-D-Pro) (Izumida et al.,1996, J. Antibiotics 49:76-80), divalent cations (e.g. Cu²⁺, Zn²⁺, andHg²⁺) (Izumida et al., 1995, J. Mar. Biotechnol. 2:163-166; Funke andSpindler, 1989, Comp. Biochem Physiol. 94B:691-695), and riboflavin andflavin derivatives (International Publication No. WO 02/23991).

Further, one of skill in the art would, when equipped with thisdisclosure and the methods exemplified herein, appreciate that achitinase-like molecule inhibitor includes such inhibitors as discoveredin the future, as can be identified by well-known criteria in the art ofpharmacology, such as the physiological results of inhibition of achitinase-like molecule as described in detail herein and/or as known inthe art. Therefore, the present invention is not limited in any way toany particular chitinase-like molecule inhibitor as exemplified ordisclosed herein; rather, the invention encompasses those inhibitorsthat would be understood by the routineer to be useful as are known inthe art and as are discovered in the future.

Further methods of identifying and producing a chitinase-like moleculeinhibitor are well known to those of ordinary skill in the art,including, but not limited, obtaining an inhibitor from a naturallyoccurring source (i.e., Streptomyces sp., Pseudomonas sp., Stylotellaaurantium). Alternatively, a chitinase-like molecule inhibitor can besynthesized chemically. Further, the routineer would appreciate, basedupon the teachings provided herein, that a chitinase-like moleculeinhibitor can be obtained from a recombinant organism. Compositions andmethods for chemically synthesizing chitinase-like molecule inhibitorsand for obtaining them from natural sources are well known in the artand are described in, among others, Yamada et al., U.S. Pat. Nos.5,413,991, and 5,070,191.

The skilled artisan would also appreciate, based on the disclosureprovided herein, that a chitinase-like molecule inhibitor encompasses anantibody that specifically binds with a chitinase-like molecule, forexample, AMCase, thereby inhibiting the action of these proteins. Forinstance, antibodies that specifically bind to YM are well known tothose of ordinary skill in the art (Webb et al., 2001, J. Biol. Chem.276:41969-41976). Similarly, antibodies to chitinase-like molecules canbe produced using standard methods disclosed herein or well known tothose of ordinary skill in the art (Harlow et al., 1988, Antibodies: ALaboratory Manual, Cold Spring Harbor, N.Y.). Thus, the presentinvention is not limited in any way to any particular antibody; instead,the invention includes any antibody that specifically binds with achitinase-like molecule either known in the art and/or identified in thefuture.

One of skill in the art will appreciate that an antibody can beadministered as a protein, a nucleic acid construct encoding a protein,or both. Numerous vectors and other compositions and methods are wellknown for administering a protein or a nucleic acid construct encoding aprotein to cells or tissues. Therefore, the invention includes a methodof administering an antibody or nucleic acid encoding an antibody (e.g.,synthetic antibody) that is specific for a chitinase-like molecule.(Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory, New York; Ausubel et al., 1997, CurrentProtocols in Molecular Biology, John Wiley & Sons, New York).

The skilled artisan would understand, based upon the disclosure providedherein, that the invention encompasses administering an antibody thatspecifically binds with a chitinase-like molecule of interest, or anucleic acid encoding the antibody, wherein the antibody moleculefurther comprises an intracellular retention sequence such that theantibody binds with the chitinase-like molecule and prevents itsexpression at the cell surface and/or its export from a cell. Suchantibodies, frequently referred to as “intrabodies”, are well known inthe art and are described in, for example, Marasco et al. (U.S. Pat. No.6,004,490) and Beerli et al. (1996, Breast Cancer Research and Treatment38:11-17). Thus, the invention encompasses methods comprising inhibitingexpression of a chitinase-like molecule on a cell and/or its secretionfrom a cell, where the skilled artisan would understand such inhibitionwould provide a benefit based upon the disclosure provided herein.

The present invention is not limited to chemical compounds andantibodies against a chitinase-like molecule. One of skill in the artwould appreciate that inhibiting the expression of a polypeptide islikewise an effective method of inhibiting the activity and function ofthe polypeptide. Thus, a method is provided for the inhibition of achitinase-like molecule by inhibiting the expression of a nucleic acidencoding a chitinase-like molecule. Methods to inhibit the expression ofa gene are well known to those of ordinary skill in the art, and includethe use of ribozymes or antisense oligonucleotide.

Antisense oligonucleotides are DNA or RNA molecules that arecomplementary to some portion of an mRNA molecule. When present in acell, antisense oligonucleotides hybridize to an existing mRNA moleculeand inhibit translation into a gene product. Inhibiting the expressionof a gene using an antisense oligonucleotide is well known in the art(Marcus-Sekura, 1988, Anal. Biochem. 172:289), as are methods ofexpressing an antisense oligonucleotide in a cell (Inoue, U.S. Pat. No.5,190,931).

Contemplated in the present invention are antisense oligonucleotidesthat are synthesized and provided to the cell by way of methods wellknown to those of ordinary skill in the art. As an example, an antisenseoligonucleotide can be synthesized to be between about 10 and about 100,more preferably between about 15 and about 50 nucleotides long. Thesynthesis of nucleic acid molecules is well known in the art, as is thesynthesis of modified antisense oligonucleotides to improve biologicalactivity in comparison to unmodified antisense oligonucleotides (Tullis,1991, U.S. Pat. No. 5,023,243).

Similarly, the expression of a gene may be inhibited by thehybridization of an antisense molecule to a promoter or other regulatoryelement of a gene, thereby affecting the transcription of the gene.Methods for the identification of a promoter or other regulatory elementthat interacts with a gene of interest are well known in the art, andinclude such methods as the yeast two hybrid system (Bartel and Fields,eds., In: The Yeast Two Hybrid System, Oxford University Press, Cary,N.C.).

Alternatively, inhibition of a gene expressing a chitinase-like moleculecan be accomplished through the use of a ribozyme. Using ribozymes forinhibiting gene expression is well known to those of skill in the art(see, e.g., Cech et al., 1992, J. Biol. Chem. 267:17479; Hampel et al.,1989, Biochemistry 28: 4929; Altman et al., U.S. Pat. No. 5,168,053).Ribozymes are catalytic RNA molecules with the ability to cleave othersingle-stranded RNA molecules. Ribozymes are known to be sequencespecific, and can therefore be modified to recognize a specificnucleotide sequence (Cech, 1988, J. Amer. Med. Assn. 260:3030), allowingthe selective cleavage of specific mRNA molecules. Given the nucleotidesequence chitinase-like molecule, one of ordinary skill in the art couldsynthesize an antisense oligonucleotide or ribozyme without undueexperimentation, provided with the disclosure and referencesincorporated herein.

One of skill in the art will appreciate that inhibitors ofchitinase-like molecule gene expression can be administered singly or inany combination thereof. Further, chitinase-like molecule inhibitors canbe administered singly or in any combination thereof in a temporalsense, in that they may be administered simultaneously, before, and/orafter each other. One of ordinary skill in the art will appreciate,based on the disclosure provided herein, that chitinase-like moleculeinhibitors to inhibit gene expression can be used to treat asthma, COPD,and other inflammatory diseases and that an inhibitor can be used aloneor in any combination with another inhibitor to effect a therapeuticresult.

B. Method of Preventing an Inflammatory Disease

It will be appreciated by one of skill in the art, when armed with thepresent disclosure including the methods detailed herein, that theinvention is not limited to treatment of an inflammatory disease oncethe disease is established. Particularly, the symptoms of the diseaseneed not have manifested to the point of detriment to the mammal;indeed, the disease need not be detected in a mammal before treatment isadministered. That is, significant pathology from an inflammatorydisease does not have to occur before the present invention may providebenefit. Therefore, the present invention, as described more fullyherein, includes a method for preventing an inflammatory disease in amammal, in that a chitinase-like molecule inhibitor, as discussedpreviously elsewhere herein, can be administered to a mammal prior tothe onset of an inflammatory disease, thereby preventing the disease asdemonstrated by the data disclosed herein.

One of skill in the art, when armed with the disclosure herein, wouldappreciate that the prevention of inflammatory disease encompassesadministering to a mammal a chitinase-like molecule inhibitor as apreventative measure against inflammatory disease. As detailed herein,the symptoms and etiologies of chitinase-like molecule-associatedinflammatory disease include tissue inflammation, increased lung volume,increased eosinophils in bronchioalveolar lavage (BAL) fluid, increasedlymphocytes in BAL fluid, increased total cells in BAL fluid, increasedalveolus size, increased deposition of crystals comprised ofchitinase-like molecules in lung tissue, increased airway resistance,increased mucus metaplasia, increased mucin expression, increasedparenchymal fibrosis, increased airway remodeling, increasedsubepithelial fibrosis, increased collagen deposition in airway tissue,epithelial hypertrophy in the lung tissue, focal organization ofcrystalline material into Masson body-like fibrotic foci, and the like.Given these etiologies and the methods disclosed elsewhere herein, theskilled artisan can recognize and prevent an inflammatory disease in amammal using a chitinase-like molecule inhibitor before the diseasepathology can be detected. This is because the data disclosed hereindemonstrate that administration of a chitinase-like molecule inhibitor,including, but not limited to, allosamidin, prevented onset of aninflammatory disease in a mammal, whether the disease was induced by anallergen (e.g. ovalbumin sensitization) or whether the mammal wasgenetically predisposed to the disease (e.g. transgenic miceconstitutively or inducibly overproducing IL-13). Accordingly, theskilled artisan would appreciate, based on the disclosure providedelsewhere herein, that the present invention includes a method ofpreventing disease comprising inhibiting a chitinase-like molecule usinga chitinase-like molecule inhibitor. Further, as more fully discussedelsewhere herein, methods of inhibiting a chitinase-like moleculeencompass a wide plethora of techniques for inhibiting not onlychitinase-like molecule activity, but also for inhibiting expression ofa nucleic acid encoding a chitinase-like molecule. Additionally, asdisclosed elsewhere herein, one skilled in the art would understand,once armed with the teaching provided herein, that the present inventionencompasses a method of preventing a wide variety of diseases whereexpression and/or activity of a chitinase-like molecule mediates thedisease. Methods for assessing whether a disease relates tooverexpression or increased activity of a chitinase-like molecule aredisclosed elsewhere herein and/or are well known in the art. Further,the invention encompasses treatment or prevention of such diseasesdiscovered in the future.

The invention further encompasses methods for treating an IL-13 mediatedinflammatory disease. This is because, as the data disclosed hereindemonstrate, IL-13 overexpression in the lungs, among other tissues,whether inducible or constitutive, mediates or is associated with theincreased expression of chitinase-like molecule in respiratory tissues,leading to, among other things, the pathologies described elsewhereherein. Thereby, the present invention includes methods of treating anIL-13 mediated inflammatory disease using the methods of the presentinvention.

The invention encompasses administration of a chitinase-like moleculeinhibitor to practice the methods of the invention; the skilled artisanwould understand, based on the disclosure provided herein, how toformulate and administer the appropriate chitinase-like moleculeinhibitor to a mammal. Indeed, the successful administration ofchitinase-like molecule inhibitors has been extensively reduced topractice as exemplified herein. However, the present invention is notlimited to any particular method of administration or treatment regimen.This is especially true where it would be appreciated by one skilled inthe art, equipped with the disclosure provided herein, including theextensive reduction to practice using an art-recognized model ofinflammatory disease, that methods of administering a chitinase-likemolecule inhibitor can be readily determined by one of skill in thepharmacological arts.

More specifically, the data disclosed herein demonstrate that increasedexpression of IL-13 mediates or is correlated with increased level of achitinase-like molecule (e.g., Ym, AMCase, and the like) and thatinhibiting a chitinase-like molecule using, among other things, anantibody that specifically binds with the chitinase-like molecule,prevents, ameliorates, and/or treats inflammatory disease. That is, forinstance, AMCase mRNA is expressed at a greater level in inflammatorydisease cells and/or tissues and administration of antibody thatspecifically binds with AMCase treats the disease in an art-recognizedanimal model of inflammatory disease. Further, the data disclosed hereindemonstrate similar results relating to expression of Ym and inhibitionof Ym using a chemical compound, i.e., allosamidin, which is a knownchitinase-like molecule inhibitor. The skilled artisan will appreciatethat the present invention is not limited to these chitinase-likemolecules or to these chitinase-like molecule inhibitors, and the datadisclosed herein amply demonstrate that inhibition of a chitinase-likemolecule can effectively treat and/or prevent an inflammatory disease.

As used herein, the term “pharmaceutically-acceptable carrier” means achemical composition with which an appropriate chitinase-like moleculeinhibitor may be combined and which, following the combination, can beused to administer the appropriate chitinase-like molecule inhibitor toa mammal.

The pharmaceutical compositions useful for practicing the invention maybe administered to deliver a dose of between about 0.1 ng/kg/day and 100mg/kg/day.

Pharmaceutical compositions that are useful in the methods of theinvention may be administered systemically in oral solid formulations,ophthalmic, suppository, aerosol, topical or other similar formulations.In addition to the appropriate chitinase-like molecule inhibitor, suchpharmaceutical compositions may contain pharmaceutically acceptablecarriers and other ingredients known to enhance and facilitate drugadministration. Other possible formulations, such as nanoparticles,liposomes, resealed erythrocytes, and immunologically based systems mayalso be used to administer an appropriate chitinase-like moleculeinhibitor according to the methods of the invention.

Compounds which are identified using any method described herein aspotential useful compounds for treatment and/or prevention of a diseaseof interest can be formulated and administered to a mammal for treatmentof the diseases disclosed herein are now described.

The invention encompasses the preparation and use of pharmaceuticalcompositions comprising a compound useful for treatment of the diseasesdisclosed herein as an active ingredient. Such a pharmaceuticalcomposition may consist of the active ingredient alone, in a formsuitable for administration to a subject, or the pharmaceuticalcomposition may comprise the active ingredient and one or morepharmaceutically acceptable carriers, one or more additionalingredients, or some combination of these. The active ingredient may bepresent in the pharmaceutical composition in the form of aphysiologically acceptable ester or salt, such as in combination with aphysiologically acceptable cation or anion, as is well known in the art.

As used herein, the term “pharmaceutically acceptable carrier” means achemical composition with which the active ingredient may be combinedand which, following the combination, can be used to administer theactive ingredient to a subject.

As used herein, the term “physiologically acceptable” ester or saltmeans an ester or salt form of the active ingredient which is compatiblewith any other ingredients of the pharmaceutical composition, which isnot deleterious to the subject to which the composition is to beadministered.

The formulations of the pharmaceutical compositions described herein maybe prepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient into association with a carrier or one ormore other accessory ingredients, and then, if necessary or desirable,shaping or packaging the product into a desired single- or multi-doseunit.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions which aresuitable for ethical administration to humans, it will be understood bythe skilled artisan that such compositions are generally suitable foradministration to animals of all sorts. Modification of pharmaceuticalcompositions suitable for administration to humans in order to renderthe compositions suitable for administration to various animals is wellunderstood, and the ordinarily skilled veterinary pharmacologist candesign and perform such modification with merely ordinary, if any,experimentation. Subjects to which administration of the pharmaceuticalcompositions of the invention is contemplated include, but are notlimited to, humans and other primates, mammals including commerciallyrelevant mammals such as cattle, pigs, horses, sheep, cats and dogs, andbirds including commercially relevant birds such as chickens, ducks,geese, and turkeys.

Pharmaceutical compositions that are useful in the methods of theinvention may be prepared, packaged, or sold in formulations suitablefor oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal,buccal, intravenous, ophthalmic, intrathecal or another route ofadministration. Other contemplated formulations include projectednanoparticles, liposomal preparations, resealed erythrocytes containingthe active ingredient, and immunologically-based formulations.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in bulk, as a single unit dose, or as a plurality of single unitdoses. As used herein, a “unit dose” is discrete amount of thepharmaceutical composition comprising a predetermined amount of theactive ingredient. The amount of the active ingredient is generallyequal to the dosage of the active ingredient which would be administeredto a subject or a convenient fraction of such a dosage such as, forexample, one-half or one-third of such a dosage.

The relative amounts of the active ingredient, the pharmaceuticallyacceptable carrier, and any additional ingredients in a pharmaceuticalcomposition of the invention will vary, depending upon the identity,size, and condition of the subject treated and further depending uponthe route by which the composition is to be administered. By way ofexample, the composition may comprise between 0.1% and 100% (w/w) activeingredient.

In addition to the active ingredient, a pharmaceutical composition ofthe invention may further comprise one or more additionalpharmaceutically active agents. Particularly contemplated additionalagents include anti-emetics and scavengers such as cyanide and cyanatescavengers.

Controlled- or sustained-release formulations of a pharmaceuticalcomposition of the invention may be made using conventional technology.

A formulation of a pharmaceutical composition of the invention suitablefor oral administration may be prepared, packaged, or sold in the formof a discrete solid dose unit including, but not limited to, a tablet, ahard or soft capsule, a cachet, a troche, or a lozenge, each containinga predetermined amount of the active ingredient. Other formulationssuitable for oral administration include, but are not limited to, apowdered or granular formulation, an aqueous or oily suspension, anaqueous or oily solution, or an emulsion.

As used herein, an “oily” liquid is one which comprises acarbon-containing liquid molecule and which exhibits a less polarcharacter than water.

A tablet comprising the active ingredient may, for example, be made bycompressing or molding the active ingredient, optionally with one ormore additional ingredients. Compressed tablets may be prepared bycompressing, in a suitable device, the active ingredient in afree-flowing form such as a powder or granular preparation, optionallymixed with one or more of a binder, a lubricant, an excipient, a surfaceactive agent, and a dispersing agent. Molded tablets may be made bymolding, in a suitable device, a mixture of the active ingredient, apharmaceutically acceptable carrier, and at least sufficient liquid tomoisten the mixture. Pharmaceutically acceptable excipients used in themanufacture of tablets include, but are not limited to, inert diluents,granulating and disintegrating agents, binding agents, and lubricatingagents. Known dispersing agents include, but are not limited to, potatostarch and sodium starch glycollate. Known surface active agentsinclude, but are not limited to, sodium lauryl sulphate. Known diluentsinclude, but are not limited to, calcium carbonate, sodium carbonate,lactose, microcrystalline cellulose, calcium phosphate, calcium hydrogenphosphate, and sodium phosphate. Known granulating and disintegratingagents include, but are not limited to, corn starch and alginic acid.Known binding agents include, but are not limited to, gelatin, acacia,pre-gelatinized maize starch, polyvinylpyrrolidone, and hydroxypropylmethylcellulose. Known lubricating agents include, but are not limitedto, magnesium stearate, stearic acid, silica, and talc.

Tablets may be non-coated or they may be coated using known methods toachieve delayed disintegration in the gastrointestinal tract of asubject, thereby providing sustained release and absorption of theactive ingredient. By way of example, a material such as glycerylmonostearate or glyceryl distearate may be used to coat tablets. Furtherby way of example, tablets may be coated using methods described in U.S.Pat. Nos. 4,256,108; 4,160,452; and 4,265,874 to formosmotically-controlled release tablets. Tablets may further comprise asweetening agent, a flavoring agent, a coloring agent, a preservative,or some combination of these in order to provide pharmaceuticallyelegant and palatable preparation.

Hard capsules comprising the active ingredient may be made using aphysiologically degradable composition, such as gelatin. Such hardcapsules comprise the active ingredient, and may further compriseadditional ingredients including, for example, an inert solid diluentsuch as calcium carbonate, calcium phosphate, or kaolin.

Soft gelatin capsules comprising the active ingredient may be made usinga physiologically degradable composition, such as gelatin. Such softcapsules comprise the active ingredient, which may be mixed with wateror an oil medium such as peanut oil, liquid paraffin, or olive oil.

Liquid formulations of a pharmaceutical composition of the inventionwhich are suitable for oral administration may be prepared, packaged,and sold either in liquid form or in the form of a dry product intendedfor reconstitution with water or another suitable vehicle prior to use.

Liquid suspensions may be prepared using conventional methods to achievesuspension of the active ingredient in an aqueous or oily vehicle.Aqueous vehicles include, for example, water and isotonic saline. Oilyvehicles include, for example, almond oil, oily esters, ethyl alcohol,vegetable oils such as arachis, olive, sesame, or coconut oil,fractionated vegetable oils, and mineral oils such as liquid paraffin.Liquid suspensions may further comprise one or more additionalingredients including, but not limited to, suspending agents, dispersingor wetting agents, emulsifying agents, demulcents, preservatives,buffers, salts, flavorings, coloring agents, and sweetening agents. Oilysuspensions may further comprise a thickening agent. Known suspendingagents include, but are not limited to, sorbitol syrup, hydrogenatededible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gumacacia, and cellulose derivatives such as sodium carboxymethylcellulose,methylcellulose, and hydroxypropylmethylcellulose. Known dispersing orwetting agents include, but are not limited to, naturally-occurringphosphatides such as lecithin, condensation products of an alkyleneoxide with a fatty acid, with a long chain aliphatic alcohol, with apartial ester derived from a fatty acid and a hexitol, or with a partialester derived from a fatty acid and a hexitol anhydride (e.g.polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylenesorbitol monooleate, and polyoxyethylene sorbitan monooleate,respectively). Known emulsifying agents include, but are not limited to,lecithin and acacia. Known preservatives include, but are not limitedto, methyl, ethyl, or n-propyl-para-hydroxybenzoates, ascorbic acid, andsorbic acid. Known sweetening agents include, for example, glycerol,propylene glycol, sorbitol, sucrose, and saccharin. Known thickeningagents for oily suspensions include, for example, beeswax, hardparaffin, and cetyl alcohol.

Liquid solutions of the active ingredient in aqueous or oily solventsmay be prepared in substantially the same manner as liquid suspensions,the primary difference being that the active ingredient is dissolved,rather than suspended in the solvent. Liquid solutions of thepharmaceutical composition of the invention may comprise each of thecomponents described with regard to liquid suspensions, it beingunderstood that suspending agents will not necessarily aid dissolutionof the active ingredient in the solvent. Aqueous solvents include, forexample, water and isotonic saline. Oily solvents include, for example,almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis,olive, sesame, or coconut oil, fractionated vegetable oils, and mineraloils such as liquid paraffin.

Powdered and granular formulations of a pharmaceutical preparation ofthe invention may be prepared using known methods. Such formulations maybe administered directly to a subject, used, for example, to formtablets, to fill capsules, or to prepare an aqueous or oily suspensionor solution by addition of an aqueous or oily vehicle thereto. Each ofthese formulations may further comprise one or more of dispersing orwetting agent, a suspending agent, and a preservative. Additionalexcipients, such as fillers and sweetening, flavoring, or coloringagents, may also be included in these formulations.

A pharmaceutical composition of the invention may also be prepared,packaged, or sold in the form of oil-in-water emulsion or a water-in-oilemulsion. The oily phase may be a vegetable oil such as olive or arachisoil, a mineral oil such as liquid paraffin, or a combination of these.Such compositions may further comprise one or more emulsifying agentssuch as naturally occurring gums such as gum acacia or gum tragacanth,naturally-occurring phosphatides such as soybean or lecithinphosphatide, esters or partial esters derived from combinations of fattyacids and hexitol anhydrides such as sorbitan monooleate, andcondensation products of such partial esters with ethylene oxide such aspolyoxyethylene sorbitan monooleate. These emulsions may also containadditional ingredients including, for example, sweetening or flavoringagents.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for rectal administration. Such acomposition may be in the form of, for example, a suppository, aretention enema preparation, and a solution for rectal or colonicirrigation.

Suppository formulations may be made by combining the active ingredientwith a non-irritating pharmaceutically acceptable excipient which issolid at ordinary room temperature (i.e. about 20° C.) and which isliquid at the rectal temperature of the subject (i.e. about 37° C. in ahealthy human). Suitable pharmaceutically acceptable excipients include,but are not limited to, cocoa butter, polyethylene glycols, and variousglycerides. Suppository formulations may further comprise variousadditional ingredients including, but not limited to, antioxidants andpreservatives.

Retention enema preparations or solutions for rectal or colonicirrigation may be made by combining the active ingredient with apharmaceutically acceptable liquid carrier. As is well known in the art,enema preparations may be administered using, and may be packagedwithin, a delivery device adapted to the rectal anatomy of the subject.Enema preparations may further comprise various additional ingredientsincluding, but not limited to, antioxidants and preservatives.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for vaginal administration. Such acomposition may be in the form of, for example, a suppository, animpregnated or coated vaginally-insertable material such as a tampon, adouche preparation, or gel or cream or a solution for vaginalirrigation.

Methods for impregnating or coating a material with a chemicalcomposition are known in the art, and include, but are not limited tomethods of depositing or binding a chemical composition onto a surface,methods of incorporating a chemical composition into the structure of amaterial during the synthesis of the material (i.e. such as with aphysiologically degradable material), and methods of absorbing anaqueous or oily solution or suspension into an absorbent material, withor without subsequent drying.

Douche preparations or solutions for vaginal irrigation may be made bycombining the active ingredient with a pharmaceutically acceptableliquid carrier. As is well known in the art, douche preparations may beadministered using, and may be packaged within, a delivery deviceadapted to the vaginal anatomy of the subject. Douche preparations mayfurther comprise various additional ingredients including, but notlimited to, antioxidants, antibiotics, antifungal agents, andpreservatives.

As used herein, “parenteral administration” of a pharmaceuticalcomposition includes any route of administration characterized byphysical breaching of a tissue of a subject and administration of thepharmaceutical composition through the breach in the tissue. Parenteraladministration thus includes, but is not limited to, administration of apharmaceutical composition by injection of the composition, byapplication of the composition through a surgical incision, byapplication of the composition through a tissue-penetrating non-surgicalwound, and the like. In particular, parenteral administration iscontemplated to include, but is not limited to, subcutaneous,intraperitoneal, intravenous, intramuscular, intracisternal injection,and kidney dialytic infusion techniques.

Formulations of a pharmaceutical composition suitable for parenteraladministration comprise the active ingredient combined with apharmaceutically acceptable carrier, such as sterile water or sterileisotonic saline. Such formulations may be prepared, packaged, or sold ina form suitable for bolus administration or for continuousadministration. Injectable formulations may be prepared, packaged, orsold in unit dosage form, such as in ampules or in multi-dose containerscontaining a preservative. Formulations for parenteral administrationinclude, but are not limited to, suspensions, solutions, emulsions inoily or aqueous vehicles, pastes, and implantable sustained-release orbiodegradable formulations. Such formulations may further comprise oneor more additional ingredients including, but not limited to,suspending, stabilizing, or dispersing agents. In one embodiment of aformulation for parenteral administration, the active ingredient isprovided in dry (i.e. powder or granular) form for reconstitution with asuitable vehicle (e.g., sterile pyrogen-free water) prior to parenteraladministration of the reconstituted composition.

The pharmaceutical compositions may be prepared, packaged, or sold inthe form of a sterile injectable aqueous or oily suspension or solution.This suspension or solution may be formulated according to the knownart, and may comprise, in addition to the active ingredient, additionalingredients such as the dispersing agents, wetting agents, or suspendingagents described herein. Such sterile injectable formulations may beprepared using a non-toxic parenterally-acceptable diluent or solvent,such as water or 1,3-butane diol, for example. Other acceptable diluentsand solvents include, but are not limited to, Ringer's solution,isotonic sodium chloride solution, and fixed oils such as syntheticmono- or di-glycerides. Other parentally-administrable formulationswhich are useful include those which comprise the active ingredient inmicrocrystalline form, in a liposomal preparation, or as a component ofa biodegradable polymer systems. Compositions for sustained release orimplantation may comprise pharmaceutically acceptable polymeric orhydrophobic materials such as an emulsion, an ion exchange resin, asparingly soluble polymer, or a sparingly soluble salt.

Formulations suitable for topical administration include, but are notlimited to, liquid or semi-liquid preparations such as liniments,lotions, oil-in-water or water-in-oil emulsions such as creams,ointments or pastes, and solutions or suspensions.Topically-administrable formulations may, for example, comprise fromabout 1% to about 10% (w/w) active ingredient, although theconcentration of the active ingredient may be as high as the solubilitylimit of the active ingredient in the solvent Formulations for topicaladministration may further comprise one or more of the additionalingredients described herein.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for pulmonary administration via thebuccal cavity. Such a formulation may comprise dry particles whichcomprise the active ingredient and which have a diameter in the rangefrom about 0.5 to about 7 nanometers, and preferably from about 1 toabout 6 nanometers. Such compositions are conveniently in the form ofdry powders for administration using a device comprising a dry powderreservoir to which a stream of propellant may be directed to dispersethe powder or using a self-propelling solvent/powder-dispensingcontainer such as a device comprising the active ingredient dissolved orsuspended in a low-boiling propellant in a sealed container. Preferably,such powders comprise particles wherein at least 98% of the particles byweight have a diameter greater than 0.5 nanometers and at least 95% ofthe particles by number have a diameter less than 7 nanometers. Morepreferably, at least 95% of the particles by weight have a diametergreater than 1 nanometer and at least 90% of the particles by numberhave a diameter less than 6 nanometers. Dry powder compositionspreferably include a solid fine powder diluent such as sugar and areconveniently provided in a unit dose form.

Low boiling propellants generally include liquid propellants having aboiling point of below 65° F. at atmospheric pressure. Generally thepropellant may constitute 50 to 99.9% (w/w) of the composition, and theactive ingredient may constitute 0.1 to 20% (w/w) of the composition.The propellant may further comprise additional ingredients such as aliquid non-ionic or solid anionic surfactant or a solid diluent(preferably having a particle size of the same order as particlescomprising the active ingredient).

Pharmaceutical compositions of the invention formulated for pulmonarydelivery may also provide the active ingredient in the form of dropletsof a solution or suspension. Such formulations may be prepared,packaged, or sold as aqueous or dilute alcoholic solutions orsuspensions, optionally sterile, comprising the active ingredient, andmay conveniently be administered using any nebulization or atomizationdevice. Such formulations may further comprise one or more additionalingredients including, but not limited to, a flavoring agent such assaccharin sodium, a volatile oil, a buffering agent, a surface activeagent, or a preservative such as methylhydroxybenzoate. The dropletsprovided by this route of administration preferably have an averagediameter in the range from about 0.1 to about 200 nanometers.

The formulations described herein as being useful for pulmonary deliveryare also useful for intranasal delivery of a pharmaceutical compositionof the invention.

Another formulation suitable for intranasal administration is a coarsepowder comprising the active ingredient and having an average particlefrom about 0.2 to 500 micrometers. Such a formulation is administered inthe manner in which snuff is taken i.e. by rapid inhalation through thenasal passage from a container of the powder held close to the nares.

Formulations suitable for nasal administration may, for example,comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) ofthe active ingredient, and may further comprise one or more of theadditional ingredients described herein.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for buccal administration. Suchformulations may, for example, be in the form of tablets or lozengesmade using conventional methods, and may, for example, contain 0.1 to20% (w/w) active ingredient, the balance comprising an orallydissolvable or degradable composition and, optionally, one or more ofthe additional ingredients described herein. Alternately, formulationssuitable for buccal administration may comprise a powder or anaerosolized or atomized solution or suspension comprising the activeingredient. Such powdered, aerosolized, or aerosolized formulations,when dispersed, preferably have an average particle or droplet size inthe range from about 0.1 to about 200 nanometers, and may furthercomprise one or more of the additional ingredients described herein.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for ophthalmic administration. Suchformulations may, for example, be in the form of eye drops including,for example, a 0.1-1.0% (w/w) solution or suspension of the activeingredient in an aqueous or oily liquid carrier. Such drops may furthercomprise buffering agents, salts, or one or more other of the additionalingredients described herein. Other opthalmically-administrableformulations which are useful include those which comprise the activeingredient in microcrystalline form or in a liposomal preparation.

As used herein, “additional ingredients” include, but are not limitedto, one or more of the following: excipients; surface active agents;dispersing agents; inert diluents; granulating and disintegratingagents; binding agents; lubricating agents; sweetening agents; flavoringagents; coloring agents; preservatives; physiologically degradablecompositions such as gelatin; aqueous vehicles and solvents; oilyvehicles and solvents; suspending agents; dispersing or wetting agents;emulsifying agents, demulcents; buffers; salts; thickening agents;fillers; emulsifying agents; antioxidants; antibiotics; antifungalagents; stabilizing agents; and pharmaceutically acceptable polymeric orhydrophobic materials. Other “additional ingredients” which may beincluded in the pharmaceutical compositions of the invention are knownin the art and described, for example in Genaro, ed., 1985, Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa., which isincorporated herein by reference.

Typically dosages of the compound of the invention which may beadministered to an animal, preferably a human, range in amount fromabout 0.01 mg to 20 about 100 g per kilogram of body weight of theanimal. While the precise dosage administered will vary depending uponany number of factors, including, but not limited to, the type of animaland type of disease state being treated, the age of the animal and theroute of administration. Preferably, the dosage of the compound willvary from about 1 mg to about 100 mg per kilogram of body weight of theanimal. More preferably, the dosage will vary from about 1 μg to about 1g per kilogram of body weight of the animal. The compound can beadministered to an animal as frequently as several times daily, or itcan be administered less frequently, such as once a day, once a week,once every two weeks, once a month, or even less frequently, such asonce every several months or even once a year or less. The frequency ofthe dose will be readily apparent to the skilled artisan and will dependupon any number of factors, such as, but not limited to, the type andseverity of the disease being treated, the type and age of the animal,etc.

C. Methods of Identifying a Useful Compound

The invention encompasses a method for identifying a compound orintervention that treats an inflammatory disease. One skilled in the artwould appreciate, based upon the disclosure provided herein, thatassessing the expression and/or activity of a chitinase-like moleculecan be performed by assessing, among other things, the levels of achitinase-like molecule or the mRNA that encodes it in a cell or tissue,and the like, and then the level can be compared to the level in anotherwise identical cell or tissue to which the compound is notadministered. Alternatively, the level of chitinase-like molecule or themRNA that encode it in a cell or tissue contacted with a compound can becompared with the level of the chitinase-like molecule or its mRNA inthe cell or tissue prior to administration of the compound. One skilledin the art would understand that such compound can be a useful potentialtherapeutic for treating and/or preventing an inflammatory disease, andfor treating and preventing an IL-13 mediated inflammatory disease,and/or for treating a disease associated with and/or mediated by a Th2inflammatory response.

The skilled artisan would further appreciate that the methods foridentifying a compound useful for inhibiting a chitinase-like moleculeinclude methods wherein a compound is administered to a cell, tissue, oranimal. That is, the skilled artisan, when armed with the presentdisclosure, would recognize that the teachings herein can be used toidentify a compound useful for inhibiting a chitinase-like molecule in acell or tissue expressing a chitinase-like molecule. Such cells andtissues are well known in the art, and can include cells and tissuesderived from a transgenic non-human animal having altered expressionIL-13, IL-4, and/or a chitinase-like molecule, or a transgenic animalcomprising an inflammatory disease, and/or a cell or tissue derivedtherefrom.

Additionally, a cell or tissue comprising expression of a chitinase-likemolecule can be contacted with a compound and the level of thechitinase-like molecule can be assessed and compared to the level of thechitinase-like molecule in the cell and/or tissue prior toadministration of the compound. Further, the level of the chitinase-likemolecule can be compared to the level of the chitinase-like molecule inan otherwise identical cell or tissue not contacted with the compound.

One skilled in the art would appreciate, based upon the disclosureprovided herein, that the cell or tissue can express endogenouschitinase-like molecule, but the invention further encompasses a cell ortissue that has been modified to express a chitinase-like molecule nototherwise expressed in the tissue, e.g., a nucleic encoding achitinase-like molecule of interest can be introduced and expressed inthe cell or tissue where it is not typically expressed, or is expressedat a different level than after the nucleic acid is introduced into thecell or tissue. Thus, the invention includes a wide plethora of assays,comprising a cell, tissue, or an animal, wherein the level of achitinase-like molecule can be assessed in the presence or absence of acompound. Accordingly, the skilled artisan would be able to identify acompound using the methods disclosed herein and cell culture and cellpropagation techniques well known in the art to assess the ability of acompound to affect the level of a chitinase-like molecule. Therefore,the present invention further encompasses a method of identifying acompound useful for inhibiting a chitinase-like molecule in a cell ortissue, as well as in an animal.

One of skill in the art would understand, based upon the disclosureprovided herein, that the invention includes a method of identifying acompound useful for treating an inflammatory disease in a mammal. Aswould be understood by one skilled in the art armed with the teachingsprovided herein, the method encompasses identifying a compound thattreats an inflammatory disease in a cell or tissue. The method comprisesidentifying a substance or compound that inhibits the expression and/oractivity of a chitinase-like molecule in a mammal (including in a cellor tissue thereof), preferably in the respiratory tract. This isbecause, as discussed elsewhere herein, the data demonstrate thatinhibiting the expression or activity of a chitinase-like moleculeprovides a therapeutic benefit thereby treating or preventing aninflammatory disease mediated by or associated with increased expressionor activity of a chitinase-like molecule. This is because the presentinvention discloses, for the first time, that increased level of achitinase-like molecule is associated with, or mediates, such diseaseand that inhibiting the chitinase-like molecule (e.g., YM, AMCase, andthe like), using a chitinase-like molecule inhibitor (e.g. allosamidin,an antibody specific for the chitinase-like molecule, such as, but notlimited to, an antibody that specifically binds with AMCase) preventsand/or treats the disease.

Thus, the skilled artisan, once armed with the teachings of theinvention, would appreciate that a compound that inhibits achitinase-like molecule is a powerful potential therapeutic orprophylactic treatment of inflammatory disease, such that identificationof such a compound identifies a potential therapeutic for such disease.

The method comprises administering to a mammal afflicted with aninflammatory disease, a compound, and comparing the level of achitinase-like molecule in the mammal before and after administration ofthe compound. The routineer would understand, based on the disclosureprovided herein, that a lower level of a chitinase-like molecule or themRNA that encodes it in the mammal after administration of the compoundcompared with the level of a chitinase-like molecule or its mRNA beforeadministration of the compound indicates that the compound is useful fortreating an inflammatory disease in a mammal.

This is because, as stated previously elsewhere herein, it has beendiscovered that inhibiting a chitinase-like molecule in an animal treatsor prevents a disease associated with increased chitinase-like moleculeexpression and/or activity, e.g., an inflammatory disease with enhancedtissue remodeling and fibrosis. The skilled artisan would alsoappreciate, in view of the disclosure provided herein, that assays todetermine the level of a chitinase-like molecule in a mammal, includinga cell or tissue thereof, include those well known in the art, or thoseto be developed in the future, all of which can be used to assess thelevel of a chitinase-like molecule in a mammal (or cell or tissuethereof) before and after administration of the compound. The skilledartisan would further appreciate that the levels of a chitinase-likemolecule, as disclosed elsewhere herein, include levels ofchitinase-like molecule activity and levels of chitinase-like moleculeexpression. Further, the invention encompasses a compound identifiedusing this method.

The invention further includes additional methods for identifying acompound useful for inhibiting a chitinase-like molecule and thereby aninflammatory disease in a mammal. More specifically, the methodcomprises assessing the level of a chitinase-like molecule expression,production, or activity in a mammal (or a cell or tissue thereof) towhich the compound is administered in comparison to an identical mammal(or cell or tissue thereof) to which the compound is not administered.Additionally, the method comprises comparing the level of achitinase-like molecule in the same mammal, or cell or tissue thereof,before and after administration of a compound of interest. A lower levelof a chitinase-like molecule expression, production, or activity in themammal administered the compound when compared to an identical mammalnot administered the compound, or to the same mammal prior toadministration of the compound, is an indication that the compound isuseful for inhibiting a chitinase like molecule which is therefore auseful potential therapeutic to treat and/or prevent inflammatorydisease in a mammal. This is because the present invention discloses,for the first time, that a chitinase-like molecule plays a clear role inthe pathology of inflammatory diseases and that inhibiting achitinase-like molecule treats and/or prevents disease in anart-recognized animal model of inflammatory disease. Clearly, asdemonstrated elsewhere herein, a compound that inhibits chitinase-likemolecules is an important potential therapeutic compound useful fortreatment and prevention of inflammatory disease as demonstrated by thedata disclosed herein.

As detailed elsewhere herein, the pathology of many inflammatorydiseases is mediated by the expression of IL-13 in an affected cell ortissue. Further, as would be appreciated by the skilled artisan equippedwith the present disclosure, the pathology of IL-13 mediatedinflammatory diseases is due, in part, to the expression ofchitinase-like molecules in an affected cell, organ or system. Themethods detailed above include mammals in which the levels of achitinase-like molecule can be readily assessed using the methodsdescribed herein. Thereby, the present invention includes mammals usefulfor identifying a compound that can be used for the treatment orprevention of inflammatory diseases. More particularly, the inventionincludes transgenic animals either constitutively or inducibleexpressing IL-13 in the respiratory tract. Based on the disclosureprovided herein, such transgenic mammals, when administered a compound,can be readily assayed for levels of a chitinase-like molecule, whetherthe assay be for chitinase-like molecule expression or chitinase-likemolecule activity. And such methods of identifying a compound useful fortreating and/or preventing inflammatory disease relating to using oftransgenic non-human mammals to assess whether the compound inhibits achitinase-like molecule are encompassed in the present invention.

II. Kits

The invention encompasses various kits relating to inhibiting chitinaselike molecules in a mammal which are useful, because, as disclosedelsewhere herein, inhibiting chitinase-like molecules provides a methodof treating or preventing inflammatory disease in a mammal. Thus, in oneaspect, the invention includes a kit for treating an inflammatorydisease in a mammal. The kit comprises an effective amount of achitinase-like molecule inhibitor. The kit further comprises anapplicator and an instructional material for the use thereof to be usedin accordance with the teachings provided herein.

The invention includes various kits which comprise a compound, such asan antibody that specifically binds a chitinase-like molecule, as wellas a nucleic acid encoding such an antibody, a nucleic acidcomplementary to a nucleic acid encoding a chitinase-like molecule butin an antisense orientation with respect to transcription, a ribozymecapable of cleaving a single-stranded chitinase-like molecule RNA, anapplicator, and instructional materials which describe use of thecompound to perform the methods of the invention. Although exemplarykits are described below, the contents of other useful kits will beapparent to the skilled artisan in light of the present disclosure. Eachof these kits is included within the invention.

In one aspect, the invention includes kits for treating or preventing aninflammatory disease and an inflammatory disease mediated by IL-13. Thekit is used pursuant to the methods disclosed in the invention. Briefly,the kit may be used to contact a mammal with a chemical compound thatinhibits chitinase-like molecules, or a nucleic acid complementary to anucleic acid encoding a chitinase-like molecule where the nucleic acidis in an antisense orientation with respect to transcription to reduceexpression of a chitinase-like molecule, or with an antibody thatspecifically binds with a chitinase-like molecule or a nucleic acidencoding the antibody, wherein the decreased expression, amount, oractivity of a chitinase-like molecule mediates an beneficial effect inthe mammal. Moreover, the kit comprises an applicator and aninstructional material for the use of the kit. These instructions simplyembody the examples provided herein.

The kit includes a pharmaceutically-acceptable carrier. The compositionis provided in an appropriate amount as set forth elsewhere herein.Further, the route of administration and the frequency of administrationare as previously set forth elsewhere herein.

EXPERIMENTAL EXAMPLES

The invention is now described with reference to the following examples.These examples are provided for the purpose of illustration only and theinvention should in no way be construed as being limited to theseexamples but rather should be construed to encompass any and allvariations which become evident as a result of the teaching providedherein.

The materials and methods used in the experiments presented in thisExample are now described.

Materials and Methods

GENERATION OF TRANSGENIC MICE: Transgenic mice constitutively expressinglung-tissue specific IL-13 were generated using the CC10-IL-13construct. The construct comprising the Clara cell 10 kDa protein (CC10)promoter, murine IL-13 cDNA, reverse tetracycline transactivator (rtTA),and human growth hormone intronic and polyadenylation sequences (hGH)was prepared as described in Zhu et al. (1999, J. Clin. Invest.103:779-788) Standard pronuclear injection was performed as described inHogan et al. (1986, Manipulating the Mouse Embryo: A Laboratory Manual,Cold Spring Harbor, N.Y.). Resultant mice were screened and founderanimals were identified using both Southern blot and PCR. The foundermice were bred onto a C57BL/6 background as described in Zhu et al.(1999, J. Clin. Invest. 103:779-788).

The generation of externally regulatable transgenic mouse systemcomprised two constructs (FIG. 5). The first construct (CC10-rtTA-hGH),as described in Zheng et al. (2000, J. Clin. Invest. 106:1081-1093),comprised the CC10 promoter, the rtTA transactivator, and the hGHintronic, nuclear localization and polyadenylation sequences. The rtTAfusion protein comprised a mutated tet operator binding protein(tet-OBP) and the herpesvirus VP-16 transactivator (Gossen et al., 1995,Science 268:1766-1769). The second construct (tet-O-CMV-IL-13),comprised a polymeric tetracycline operator (tet-O), minimalcytomegalovirus (CMV) promoter, IL-13 cDNA, and hGH intronic,polyadenylation and nuclear localization signals, was prepared asdescribed in Ray et al. and Zheng et al. (1997, J. Clin. Invest.100:2501-2511 and 2000, J. Clin. Invest. 106:1081-1093). Transgenic micewere prepared by simultaneous microinjection of constructs into oocytes,as described in Hogan et al. (1986, Manipulating the Mouse Embryo: ALaboratory Manual, Cold Spring Harbor, N.Y.). Mice were screened by PCRand Southern blot from tail biopsy DNA as described in Zhu et al. andZheng et al. (1999, J. Clin. Invest., 103:779-788). Four founder micewere then bred with C57BL/6 mice to create transgenic mice withinducible IL-13 expression in the lungs.

Generation and Administration of Anti-AMCase Antibodies

Polyclonal antibodies to AMCase were generated by immunizing rabbitswith a peptide derived from AMCase (ADKADGLYPVADDRNAFWQ; SEQ ID NO: 13)using methods well known in the art and described in, for example,Harlow et al. (1989, Antibodies: A Laboratory Manual, Cold SpringHarbor, N.Y.).

Wild type mice were sensitized to OVA and challenged with OVA on threesuccessive days as described elsewhere herein. Sensitized mice wereadministered 0.5 ml of anti-AMCase antibodies or control serumintraperitoneally every other day starting the day before the firstaerosol exposure.

HISTOLOGIC ANALYSIS: Mice were sacrificed by cervical dislocation and amedian sternotomy was performed. The right heart was perfused withcalcium and magnesium free phosphate buffered saline (PBS). The heartand lungs were removed en bloc, and the lungs were fixed to 25 cmpressure with neutral buffered 10% formalin. They were then fixedovernight in 10% formalin, embedded in paraffin, sectioned at 5 pm, andstained. Hematoxylin and eosin (H & E), Mallory's trichrome, periodicacid-Schiff with diastase (D-PAS), alcian blue at pH 2.5, PAS/alcianblue, modified Congo red, and Papanicolau stains were used forhistological analysis.

Hydroxyproline Assays

Total lung collagen was determined by analysis of hydroxyprolinecontent. Briefly, lungs were harvested on specified times andhomogenized in 2 ml of PBS, pH 7.4, with a Tissue Tearor(PRO-Scientific, Monroe, Conn.). One-half milliliter of each sample(both lungs) was then digested in 1 ml of 6 N HCl for 8 hours at 120° C.Five microliters of citrate/acetate buffer (5% citric acid, 7.24% sodiumacetate, 3.4% sodium hydroxide, and 1.2% glacial acetic acid, pH 6.0)and 100 μl of chloramine-T solution (282 mg of chloramine-T, 2 ml ofn-propanol, 2 ml of H2O, and 16 ml of citrate/acetate buffer) were addedto 5 μl of sample, and the samples were left at room temperature for 20minutes. Next, 100 μl of Ehrlich's solution (2.5 g of 4-(dimethylamino)benzaldehyde (Aldrich, Milwaukee, Wis.), 9.3 ml of n-propanol, and 3.9ml of 70% perchloric acid (Eastman Kodak, Rochester, N.Y.) were added toeach sample, and the samples were incubated for 15 minutes at 65° C.Samples were cooled for 10 minutes and read at 550 nm on a Beckman DU640 spectrophotometer (Fullerton, Calif.). Hydroxyproline (Sigma, St.Louis, Mo.) concentrations from 0-10 μg/ml were used to construct astandard curve. (Keane et al. J. Immunol. 1999, 163:5686-82.)

BRONCHOALVEOLAR LAVAGE (BAL) AND QUANTIFICATION OF IL-13 LEVELS: Micewere killed by cervical dislocation and a median sternotomy wasperformed. The trachea was isolated by blunt dissection and smallcaliber tubing was inserted and secured in his airway. Three successivevolumes of 0.75 ml of PBS with 0.1% bovine serum albumin (BSA) wereinstilled and gently aspirated and pooled. Each BAL sample wascentrifuged and the supernatants were stored at −70° C. Cell numberswere assessed with hemocytometer and cellular differential counts wereundertaken on cytospin preparations. IL-13 levels were determined byELISA using a commercial kit according to the manufacturer'sinstructions (R&D Systems, Minneapolis, Minn.).

PHYSIOLOGICAL AIRWAY ASSESSMENT ASSAYS: Age- and gender-matchedlittermates were evaluated by both invasive and non-invasivephysiological assessment techniques.

The non-invasive techniques were used to determine the baseline airwaysresistance and the level of airways hyperresponsiveness (AHR) inunrestrained, conscious mice. That is, animals were assessed usingbarometric plethysmography using whole body plethysmography (BuxcoElectronics Inc., Troy, N.Y.) as described in Hamelmann et al. (1997,Am. J. Resp. Crit. Care Med. 156:766-775) and Kline et al. (1998, J.Immunol. 160:2555-2559). Briefly, mice were placed into whole bodyplethysmographs interfaced with computers using different pressuretransducers. Tidal volume, respiratory rate, and enhanced pause(P_(enh)) measurements were made. Airway resistance is expressed asP_(enh)=[(T_(e)/0.3T_(r))−1]×[2 P_(ef)/3 P_(if)], where P_(enh)=enhancedpause, T_(e)=expiratory time in seconds, T_(r)=relaxation time inseconds, P_(ef)=peak expiratory flow (ml), and P_(if)=peak inspiratoryflow (ml/s). Increasing doses of methacholine (Sigma Chemical Company,St. Louis, Mo.) were administered using a nebulizer for 120 seconds, andP_(enh) was determined over the following five minutes.

Invasive physiological assessments were performed in anesthetized(pentobarbital, 90 mg/kg) and tracheostomized (18 gauge angiocatheter)age- and sex-matched mice. The changes in the lung volume of the micewere measured plethysmographically by determining the pressure in aPlexiglass chamber using an inline microswitch pressure transducer. Flowwas measured by the difference between the volume signal and thetranspulmonary pressure as determined by a second microswitch pressuretransducer placed in line with the plethysmograph and animal ventilator.Resistance (with resistance due to the tracheostomy catheter eliminated)was measured using the method of Amdur and Mead (1958, Am. J. Physiol.192:364-368). Baseline measurements of pulmonary resistance wereobtained by ventilating the mouse at a volume 0.4 ml and a rate of 150breaths per minute. Increasing concentrations of methacholine in PBSwere administered by nebulization (20 one-ml breaths) using a DevilbissAerosonic nebulizer (Model 5000, Devilbiss Health Care, Somerset, Pa.)that produces particles of about 1-3 μm in diameter. Pulmonaryresistance was calculated precisely 1 minute later. Stepwise increasesin methacholine dose were then administered until the pulmonaryresistance, in comparison with the baseline level, had at least doubled.All animals received serial threefold increases in methacholine from 1to 100 mg/ml. The data are expressed as the PC₁₀₀ (provocative challenge100), which is the dose at which pulmonary resistance was 100% above thebaseline level as calculated by linear regression analysis.

LUNG VOLUME ASSESSMENT: Lung volume assessment was performed exactly asdescribed in Zheng et al. (2000, J. Clin. Invest. 106:1081-1093)

DOXYCYCLINE ADMINISTRATION: All inducible transgenic mice weremaintained on normal water until transgene activation was desired.Doxycycline (dox) was administered in drinking water (0.5 mg/ml). Doxcontaining water bottles were wrapped in aluminum foil to preventlight-induced dox breakdown.

MRNA ANALYSIS: mRNA levels were assessed using Northern blot andreverse-transcriptase polymerase chain reaction (RT-PCR). Total cellularRNA was extracted from mouse tissue using TRIZOL™ (Invitrogen, Carlsbad,Calif.) per the manufacturer's instructions. Primers specific for YM(YM-1 forward primer: TGGAATTGGTGCCCCTACAA; SEQ ID NO:1, YM-1 reverseprimer: AACTTGCACTGTGTATATTG; SEQ ID NO:2, YM-2 forward primer:AACCTCAGACATTCATTA; SEQ ID NO:3, YM-2 reverse primer:TGGTCCTTCCAGTAGGTAATA; SEQ ID NO:4, YM-3 forward primer:TATAAATCTCCATTTGACAC; SEQ ID NO:5, YM-3 reverse primer:CCTAATTTATTGTCCTTGAC; SEQ ID NO:6) and AMCase (AMCase forward primer:ATCTGCAGTGGACACACCTTCATCCTGA; SEQ ID NO:7, AMCase reverse primer:ATGAATTCAACAAGCCCTGCTTGACAAT; SEQ ID NO:8) were used in RT-PCR toamplify and detect these transcripts. Reverse transcription and PCR wereperformed using the Access RT-PCR kit from Promega (Madison, Wis.) perthe manufacturer's instructions.

IN SITU HYBRIDIZATION OF MURINE LUNGS: In situ hybridization was used tolocalize expression of both YM and AMCase in transgenic animals. Lungtissues were fixed in formaldehyde and processed into paraffin. Fivemicron sections were cut, deparaffinized, and treated with proteinase K(20 μg/ml, 37° C., 20 min). Tissues were then treated with 0.1 Mtriethylnolamine/0.25% acetic anhydride (pH 8) for 10 min at roomtemperature and rinsed in PBS. Antisense and sense probes for YM (YMantisense probe: TCCTCGAGACCCAGGGTACTGC; SEQ ID NO:9, YM sense probe:TATCTAGAGGATCTTCCTACCAGC; SEQ ID NO:10) and AMCase (AMCase antisenseprobe: TCGCTCGAGAACAAGCCCTGCTTGACAAT; SEQ ID NO:11, AMCase sense probe:GCTCTAGATGGACACACCTTCATCCTGA; SEQ ID NO:12 were generated by cloning afragment of mouse AMCase cDNA or YM cDNA into vector pBS II KS with T3and T7 primer sequences flanking the multiple cloning sites (Stratagene,La Jolla, Calif.). The oligonucleotide primers with XbaI and XhoIrestriction enzyme sites incorporated, were used to amplify DNAfragments from total lung RNA of an IL-13 transgene (+) mouse. TheRT-PCR products were digested with XbaI and XhoI and cloned into thevector pBS II KS. Sense and antisense RNA probes were generated, labeledwith a digoxigenin RNA labeling kit (Roche, Indianapolis, Ind.),denatured at 65° C., and added to commercially available hybridizationbuffer (Ambion, Austin, Tex.) at 6 ng/μl, and the hybridization mixturewas incubated with tissue overnight at 52° C. The tissues were thenwashed twice with 4×SSC for 5 min at room temperature, twice with 2×SSCfor 10 min at 37° C., and incubated with RNase A (10 μg/ml) for 45 minat 37° C. This was followed by two 10-min washes in 2×SSC at roomtemperature and three 20-minwashes in 0.2×SSC at 50° C. Probes weredetected by overnight incubation with sheep antibodies (Abs) todigoxigenin labeled with alkaline phosphatase (Roche) followed by4-nitroblue tetrazolium chloride/5-bromo-4-chloro-3-indoylphosphate, asdescribed by the manufacturer.

CRYSTAL PURIFICATION AND ANALYSIS: Crystals were purified using aficoll-gradient washing procedure as described by Guo et al. (2000, J.Biol. Chem. 275:8032-8037). Briefly, BAL fluid from IL-13 transgenicmice was loaded, in a ratio of 1:5, on top of Histopaque-1119 with adensity of 1.119 grams/ml (Sigma, St. Louis, Mo.) and centrifuged at250×g for 10 minutes at 4° C. The supernatant was removed and the pelletwas resuspended in PBS and centrifuged twice more as above. Theresulting pellet was dissolved in SDS-PAGE sample buffer and boiled for10 minutes before electrophoresis. SDS-polyacrylamide gelelectrophoresis was performed under reducing conditions usingTris-glycine 4-20% gradient gels (BioRad, Hercules, Calif.). Proteinbands were visualized by staining with Coomassie Blue, excised with ascalpel and subjected to in-gel tryptic digestion before massspectrometric analysis. The Coomassie Blue stained protein band around40 kDa was excised and washed with 50 mM ammonium bicarbonate, 50%acetonitrile for 30 minutes, followed by a 10 mM ammonium bicarbonate,50% acetonitrile wash for an additional 30 minutes. After washing, thegel pieces were dried and re-hydrated with 0.1 mg modified trypsin(Promega, Madison, Wis.) in 15 μl 10 mM ammonium bicarbonate. Digestionwas done at 37° C. for 24 hours. Matrix assisted laser desorptionionization mass spectrometry (MALDI-MS) was carried out on 1.0 μl (<5%)of the digest using a Micromass TofSpec SE mass spectrometer (Micromass,Beverly, Mass.) in reflectron mode. Prior to MALDI-MS the sample wasmixed with 1.0 μl of alpha-cyano-4-hydroxy cinnamic acid matrix solution(4.5 mg/ml in 0.05% trifluoroacetic acid, 50% acetonitrile) plus 1 μl ofinternal calibrants and then spotted onto a new single use target. Thesamples were then allowed to air dry at room temperature. The internalcalibrants used were 50 femtomoles bradykinin (monoisotopic M+H is1060.57) and 125 fmols ACTH Clip 18-39 (monoisotopic M+H is 2465.20). Atotal of 92 peptide masses (monoisotopic) were submitted for peptidemass database searching using Peptide Search (non-redundant database atthe EMBL) and ProFound (at Rockefeller University for non-redundantdatabase at NCBI). By using either algorithm, 24 of the 92 peptidemasses matched mouse chitinase 3-like 3 protein (also called YM-1 andECFL-precursor) with a minimum coverage of 59%. A subsequent searchusing unmatched peptide masses of the first pass did not yield anymeaningful matches.

CHITINASE ACTIVITY ASSAYS: BAL fluid, collected as previously described,was used in a chitinase activity assay. The chitinase activity in BALwas assessed using a fluorescence assay. Fluorogenic4-methylumbelliferyl β-D-N,N′-diacetylchitobioside was used as asubstrate. Assays were performed as the following. BAL samples wereincubated with the substrate at a concentration of 0.02 M incitrate/phosphate buffer (0.1 M/0.2 M), pH 5.2. After 15 min at 37° C.,the reaction was stopped by adding 1 ml of 0.3 M glycine/NaOH buffer, pH10.6 and the fluorescent 4-methylumbelliferone was determined with afluorimeter at excitation of 350 nm and emission 450 nm. A standardcurve was generated using 4-methylumbelliferone (Sigma). Chitinaseextract from Serratia marcescens was used as a positive control (Sigma).

AEROALLERGEN OVALBUMIN (OVA) SENSITIZATION AND CHALLENGE TESTS: OVAsensitization and challenge were accomplished using modifications of theprotocols previously described by Yang et al. (1998, J. Exp. Med.188:1739-1750). Briefly, wild type mice received intraperitoneal (i.p.)injections containing 20 μg of avian OVA (Sigma) complexed to alum(Resorptar; Indergen, New York, N.Y.). This process was repeated 5 dayslater. After an additional 7 days, animals received aerosol challengewith OVA (1% w/v) in endotoxin-free PBS or the animals receivedendotoxin-free PBS alone. The aerosol challenge was accomplished in aclosed 27×20×10 cm plastic aerosol chamber in which the mouse was placedfor 40 minutes. The aerosol was generated using an Omron NE-U07ultrasonic nebulizer (Omron Healthcare, Vernon Hills, Ill.). Mice weresacrificed twenty-four hours, forty-eight hours, and seven days afterOVA challenge.

RELATIVE INDUCTION OF YM AND AMCASE: Affymetrix murine GENE CHIP arrays(Santa Clara, Calif.) comprising 12,200 oligonucleotides were used toanalyze IL-13 induced gene expression in the murine lung. The levels ofgene expression in IL-13 expressing, dox-inducible and control mice wereanalyzed. The expression levels were standardized using housekeepinggenes (e.g. actin, GAPDH, hexokinase and the like), and a stimulationindex was calculated by dividing the target ratio in transgene (+)animals by the target ratio in transgene (−) animals. Similar geneexpression studies were performed in mice constitutively expressingIL-13 and controls. GENE CHIP assays were performed as follows. TotalRNA was isolated from the lungs of IL-13 transgenic mice and littermatenegative controls with Trizol reagent (Life Technologies, Gaithersburg,Md.). To prepare samples for Affymetrix GENECHIP analysis, cDNA wasgenerated from 15 μg of total RNA by use of a modified oligo-dT primerand a 5′ T7 15 RNA polymerase promoter oligo primer with the SuperscriptChoice System for cDNA Synthesis (Life Technologies). Afterphenol-chloroform extraction and ethanol precipitation, one-half of thecDNA reaction (0.5-1.0 μg) was used as a template for an in vitrotranscription reaction with biotinylated UTP and CTP (BioArray HighYield kit, Enzo Biochem, Farmingdale, N.Y.) by following themanufacturer's protocol. The resulting cRNA was purified on an affinityresin column (RNeasy, Qiagen, Valencia, Calif.) and quantified byultraviolet (UV) absorbance. For each reaction, 15 μg of biotinylatedcRNA were randomly fragmented to an average size of 50 nucleotides byincubating them at 94° C. for 35 min in 40 mM Tris-acetate, pH 8.1,1,000 mM potassium acetate, and 30 mM magnesium acetate. The fragmentedcRNA was divided into two aliquots that were each used for hybridizationto an MullK Affymetrix GENECHIP according to the manufacturer's protocol(Affymetrix, Santa Clara, Calif.), with a duplicate data set generatedfor all samples. Each target array was washed and scanned(Hewlett-Packard, GeneArray Scanner G2500A). Data were analyzed with theAffymetrix GENECHIP software algorithm to generate “average difference”and/or degree of difference after the values were normalized. The valuesobtained from wild type C57BL/6 littermate controls were used asbaseline and the values from IL-13 transgenic mice were expressed asrelative fold-increases.

RIBONUCLEASE PROTECTION ASSAY: Ribonuclease protection assays wereperformed using the mCK-1 template kit (PharMingen San Diego, Calif.).Ribonuclease protection assays were performed as described in, forexample, Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory, New York) and Ausubel et al., 1997(Current Protocols in Molecular Biology, John Wiley & Sons, New York).

ALLOSAMIDIN ADMINISTRATION: Allosamidin (Eli Lilly and Co., Greenfield,Ind. and Industrial Research Limited, Lower Hutt, New Zealand) wasadministered to inducible IL-13 overexpressing mice and OVA exposed wildtype mice. Mice were given 0.1 mg/kg to 10 mg/kg allosamidin i.p. orvehicle control (PBS). The animals were then sacrificed and BAL fluidanalysis, histologic and morphometric analysis, and lung volumeassessment were performed as described elsewhere herein.

The results of the experiments presented in this Example are nowdescribed.

TRANSGENIC MICE CONSTITUTIVELY EXPRESSING IL-13: Transgene (+) miceexpressing IL-13 constitutively exhibited high levels of IL-13 in BALfluid (up to 2.1 ng/ml) and detectable IL-13 mRNA in the lungs. IL-13mRNA could not be detected in the skin and other visceral organs of thetransgenic mice, indicating lung-specific expression of IL-13. Trangene(−) mice did not demonstrate detectable levels of IL-13 or IL-13 mRNA inBAL fluid or in the lungs.

Histologic analysis of transgene (+) mice demonstrated multipleasthma-like features. These features include eosinophil, lymphocyte, andmacrophage-rich inflammatory responses around the small and largeairways and in the adjoining parenchyma. This response was milder inyoung animals, or animals with low levels of BAL IL-13, and moreprominent in older animals or those with higher BAL levels of IL-13.Epithelial hypertrophy was evident in the conducting and small airwaysas well (FIG. 2).

Constitutive expression of lung-specific IL-13 also resulted inCOPD-like alveolar enlargement and wall rupture, as well as focalorganization of crystalline material into Masson body-like fibroticfoci. Further, long, thin, needlelike crystals were seen in themacrophages, alveoli, and occasionally, the airways of transgene (+)animals.

As mucus metaplasia and enhanced mucin gene expression arecharacteristic of both asthma and COPD, the effect of constitutive IL-13expression on airway mucus was undertaken. Both PAS and alcian bluestaining demonstrated that mucus accumulation was prominent in theairways of transgene (+) mice, but not in transgene (−) littermates(FIG. 3). Impressive increases in the mucin genes MUC5AC, MUC2 and MUC4mRNA were also evident in transgene (+) mice.

Airway remodeling with subepithelial fibrosis is a well documentedfeature of the asthmatic airway, and disordered repair and parenchymalfibrosis are often noted as aspects of emphysema. These features ofinflammatory diseases are associated with increased collagen deposition.Accordingly, Masson's trichrome stains, sirius red, and hydroxyprolineassays were used to evaluate the collagen deposition in the airways oftransgene (+) and (−) animals.

A small amount of collagen was seen in and near the airway wall intransgene (−) animals, and loosely packed collagen was detected in thebronchovascular bundles. In sharp contrast, enhanced collagen depositionwas seen in the subepithelial region and aventitia of the small andlarge airways of transgene (+) animals (FIG. 4), similar to findings inhuman airway disorders. Scarring and parenchymal fibrosis was observedin older animals, and these features increased with age. Increasedlevels of hydroxyproline could be detected as early as 4-6 weeks afterIL-13 production, and three month old animals had significantly (4.1fold, p<0.001) higher hydroxyproline levels than transgene (−) animals.

Both asthma and COPD patients demonstrate airway obstruction and AHR (anexaggerated bronchospastic response to non-specific agonists likemethacholine). Accordingly, studies were undertaken to determine ifthese airway alterations were present in the IL-13 transgenic animalmodel. Baseline airway resistance was mildly elevated in transgene (+)animals. In addition, AHR was also seen after methacholine challenge, asdetermined using invasive and non-invasive assessment methodologies.These data indicate asthma- and COPD-like physiological alterations arepresent in the IL-13 transgenic model.

INDUCIBLE TRANSGENIC MICE: The lung-specific inducible transgenic animalsystem allows for temporal control of IL-13 expression in the murinelungs. This mimics the waxing and waning patterns of IL-13 expressionseen in asthma and COPD and circumvents the abnormalities caused by thein utero or neonatal gene expression seen with other transgenic models.

Inducible transgenic mice were kept on normal (dox-free) water until onemonth of age. IL-13 was not detected in the BAL fluid from transgene (−)animals on dox or normal water. In the absence of dox, levels of BALIL-13≦75 pg/ml were found in transgene (+) animals. Within 24 hours ofdox administration, transgene (+) animals demonstrated increased BALIL-13 levels, and steady state levels ranging from about 0.5 to 1.5ng/ml were observed within 96 hours after dox administration. BAL IL-13levels returned to background levels within 96 hours after doxadministration ceased. IL-13 mRNA was only detectable in pulmonarytissues of transgene (+) animals.

H&E and trichrome staining demonstrated that lungs obtained fromtransgene (−) mice given normal water or dox-containing water did notdemonstrate any histological abnormalities, nor could those lungs bedistinguished from the lungs obtained from transgene (+) mice givennormal water. However, transgene (+) mice given normal water did showmild mucus metaplasia after D-PAS staining.

Conversely, transgene (+) mice given dox water exhibited notableinflammatory, mucus, and structural alterations. As little as seven daysafter dox administration, inflammation in BAL fluid was prominent. Atthis time point, there was a 7.5 fold increase in cell recovery from BALfluid and a significant increase in the percentage of BAL fluideosinophils (63%, p<0.001) in transgene (+) mice given dox water.Lymphocyte and macrophage recovery were also significantly increased(p<0.01) in these mice. Also, mononuclear, lymphocytic and eosinophilicinfiltrates were prominent in airway and peribroncheal structures, aswas an increase in mucus metaplasia. Additionally, substantial increasesin MUC-5AC, MUC-2, and MUC-4 mRNA were noted. Chronic administration ofdox resulted in subepithelial fibrosis, alveolar enlargement, andcrystal deposition, very similar to that observed in transgenic miceconstitutively expressing IL-13.

The emphysema in COPD is defined pathologically as the abnormalenlargement of the airspaces distal to the terminal bronchial of thelung (Senior and Shapiro, 1998, Fishman's Pulmonary Diseases andDisorders, Vol. 1, McGraw-Hill, N.Y.). As previously noted, miceexpressing IL-13 constitutively displayed enlarged alveoli. To determinewhether this enlargement was due to faulty development or to thedestruction of lung tissue in a normally formed lung, inducibletransgenic (+) mice were given dox water only after full lungdevelopment was completed. After dox administration, IL-13 inducedalveolar enlargement was apparent using both histologic and morphometrictechniques. In the absence of dox, normal alveoli were seen in bothtransgene (−) and (+) animals (FIG. 6).

YM CRYSTAL DEPOSITION IN IL-13 OVEREXPRESSING MICE: As previously noted,crystals were seen in both inducible and constitutive IL-13 transgenemice. The presence of the crystals was both dose- and time-dependent. Inconstitutive IL-13 mice, crystals could be seen at the earliest timepoint assessed (1 month), and impressive crystal accumulation wasevident in three month old animals. Similarly, the inducible IL-13 micedisplayed crystals in various tissues and cells of the airway at aboutthe same time intervals after dox administration. In young mice,crystals were most commonly seen in macrophages, parenchyma and alveoli,and less commonly in distal airways. In older animals, considerablealveolar and parenchymal crystal deposition was noted, including manyalveoli completely filled with crystalline deposits. Crystals weremulti-faceted, often needle shaped, and approximately 20-120 μm inlength (FIG. 7).

Crystals were purified from BAL fluid from constitutive IL-13 transgenic(+) mice using a ficoll density gradient method as described elsewhereherein. These crystals were assayed and determined to be comprised of YMproteins. No other peptides were found in the sample, and YM proteinswere not detected in transgene (−) animals, indicating that the crystalsin IL-13 transgene (+) animals comprise YM proteins.

YM GENE EXPRESSION IN IL-13 OVEREXPRESSING MICE: RT-PCR was performed asdescribed herein to determine if YM protein expression was induced byIL-13. In whole lung RNA from transgene (−) animals, YM mRNA was at ornear the lower sensitivity limits of the assay (FIG. 9). In starkcontrast, in transgenic mice constitutively expressing IL-13, YM mRNAwas detected in all time points assessed (1 to 3 month old mice).Inducible transgene (+) mice that did not receive dox demonstrated lowlevels of YM mRNA, indicating a mildly “leaky” system. Uponadministration of dox, striking increases in YM mRNA were observed aslittle as 48 hours after dox introduction, and high levels of YM mRNAexpression continued throughout the three month period in which dox wasadministered (FIG. 10).

In situ hybridization was used to localize the sites of YM proteinproduction in IL-13 transgenic animals. YM mRNA could not be detected intransgene (−) mice, but impressive levels were detected in transgene (+)animals using antisense probes. In transgene (+) animals, YM localizedintensely to macrophages and to airway epithelial cells. It should benoted that YM staining was not detected when the same tissues wereprobed using sense oligonucleotides (FIG. 11), confirming thespecificity of these results.

To ascertain whether cytokine induction of YM mRNA expression was IL-13specific, RT-PCR analysis was undertaken using whole lung RNA from avariety of other transgenic mice. IL-4 transgenic mice expressedexaggerated levels of YM in their lungs, similar to IL-13 transgenicmice. This coincides with evidence indicating that IL-4 is anotherimportant cytokine in human airway disorders, as stated previouslyelsewhere herein. Transgenic mice constitutively expressing IL-6, IL-11,vascular endothelial growth factor₁₆₅ (VEGF) and IL-10 demonstrated YMmRNA levels comparable to that of IL-13 transgene (−) littermatecontrols, further confirming that the cytokines postulated to play arole in Th2 dominated respiratory inflammation, i.e., IL-13 and IL-4,are also potent and specific inducers of YM expression.

CHITINASE ACTIVITY IN BAL FLUID FROM IL-13 OVEREXPRESSING MICE:Chitinase activity in BAL fluids from transgene (+) and (−) miceconstitutively expressing IL-13 was assayed using methods describedelsewhere herein. BAL fluid obtained from transgene (−) mice had levelsof chitinase activity ≦75 units/ml. In contrast, an impressive increasein chitinase activity in transgene (+) mice was detected (FIG. 12). Thatis, chitinase activity was detected in 1 month old transgene (+)animals, and continued to increase as the animals aged. Similarly,chitinase activity was detected in inducible transgene (+) animals aslittle as two days after dox administration, and this activity alsoincreased over time. Transgenic mice overexpressing IL-4 alsodemonstrated increased levels of chitinase activity. However, BAL fluidfrom IL-6, IL-11, VEGF, and IL-10 transgene (+) animals had basal levelsof chitinase activity. These data indicate that increased chitinaseactivity correlates with increased IL-13 and/or IL-4 expression and withincreased YM protein expression and crystal deposition in lung tissues.

AMCASE EXPRESSION IN IL-13 TRANSGENIC MICE: Conflicting reports haveindicated that YM may or may not have chitinase activity. For instance,both purified and recombinant YM have failed to demonstrate chitinaseactivity in a number of assays, indicating that it is not a chitinase,but rather a chitinase-like molecule (Chang et al., 2001, J. Biol. Chem.276:17497-17506). BAL fluid from IL-13 transgene (+) mice demonstratesdetectable chitinase activity, indicating that a chitinase familyprotein may be present in the BAL fluid of these mice. To date, AMCaseis the only enzyme identified in murine systems that demonstrates truechitinase activity. To determine whether AMCase expression was augmentedin the IL-13 transgene (+) mouse lung, RT-PCR primers specific forAMCase were used, as described above. mRNA levels in transgene (−)littermate controls were near or below detection levels in the assayemployed. Conversely, impressive increases in AMCase mRNA levels werefound in both constitutive and inducible IL-13 transgenic models. In theformer it was noted in animals that were one month to three months ofage. In the latter as little as seven days after dox administration,respectively. Similar to YM protein expression, AMCase expression wasspecific to IL-13 transgenic animals, as it was not detected in lungsfrom the other transgenic animals (IL-10, VEGF, IL-6, and IL-11)assessed herein.

In situ hybridization was employed to localize AMCase production inIL-13 transgenic mice. Prominent accumulation of AMCase mRNA wasdetected in epithelial cells and, to a lesser extent, in themacrophages. In contrast, AMCase mRNA was not detected in transgene (−)mice (FIG. 14).

CHITINASE GENE EXPRESSION IN AN OVA-INDUCED MURINE ASTHMA MODEL: Studieswere performed to ascertain whether YM proteins and/or AMCase wereinduced in the standard Th2-driven murine asthma model. To this end, thechitinase activity of BAL fluid and the expression of these two genes inlungs of OVA sensitized wild type mice was investigated. In animals thatwere OVA sensitized, but did not receive an OVA challenge, mRNA levelsand BAL fluid chitinase activity were near the limits of detectionlevels of the assays. However, after OVA challenge, YM and AMCase mRNAlevels were readily detected and persisted for 7 days after antigenchallenge. BAL fluid chitinase activity increased accordingly as well(FIG. 15).

RELATIVE INDUCTION OF YM AND AMCASE: Affymetrix GENE CHIPS, comprising12,200 oligonucleotides, were used to assess the IL-13 induced geneexpression alterations in inducible transgenic animals that had beengiven dox from 1 month to 2 months of age, and compared with transgene(−) littermate controls. The GENE CHIP analysis indicated that over 200genes were increased by at least 2.5 fold, and approximately 140 geneswere downregulated by dox administration. Importantly, the gene mostprominently induced after dox administration was YM (stimulation indexof 64.1±0.5). AMCase was not present on the chip, but another chitinasefamily gene, BRP39, was the twelfth most prominently induced gene in thearray (stimulation index of 7.1±0.5). It should be noted that doxadministration did not cause significant alterations in gene expressionof transgene (−) animals.

As AMCase was not present on the GENE CHIP, other methods, includingRT-PCR, Northern blot analysis, and ribonuclease protection assays wereemployed. All indicated that AMCase is an important downstream target ofIL-13. This further demonstrates that AMCase is induced during thecourse of and potentially involved in the pathogenesis of IL-13 inducedrespiratory inflammation.

EFFECTS OF ALLOSAMIDIN IN MURINE ASTHMA MODELS: Allosamidin is a knownpotent and selective inhibitor of chitinases. Wild type mice weresensitized to OVA and challenged with OVA a number of days later. Oneday prior to OVA challenge, mice were randomized into two groups andreceived daily i.p. doses of allosamidin or of vehicle control. Similarto the symptoms of human asthma, the OVA sensitization and challengemodel results in a brisk eosinophil- and lymphocyte-rich inflammatoryresponse with prominent mucus metaplasia and goblet cell hyperplasia.Allosamidin administration resulted in a dose-dependent inhibition oftotal cell, eosinophil and lymphocyte influx in the OVA challenged lung(FIG. 16). The inhibitory effect was most prominent in the highest dosetested (10 mg/kg), and was still prominent at the lowest (1 mg/kg).Therefore, allosamidin inhibited antigen-induced inflammation in anart-recognized murine model of human asthma.

Similarly, six-week old transgene (+) IL-13 mice were randomized toreceive fourteen daily doses i.p. of allosamidin (1 mg/kg) or vehiclecontrol. The animals were then sacrificed and the IL-13 phenotype wasevaluated. In comparison to transgene (+) mice that received the vehiclecontrol, transgene (+) mice that received allosamidin injectionsdisplayed markedly decreased BAL fluid cell recovery, decreased tissueinflammation, and impressive decreases in BAL and tissue eosinophils andlymphocytes. Morphometric, histologic, and lung volume assessmentanalysis indicated that the allosamidin treated animals had decreasedlung volumes and smaller alveoli than the littermate controls,indicating that allosamidin is a potent inhibitor of both IL-13 inducedinflammation and lung remodeling. It is important to note that in theseexperiments, allosamidin administration commenced six weeks after thebirth of the transgenic mice constitutively expressing IL-13, that is,after the asthma-like phenotype had commenced. Thus allosamidintreatment in these mice and COPD decreased the progression of lungpathology even after the pathologic response had been initiated. This isanalogous to therapeutic situations in humans where pharmaceuticalcompositions are administered after respiratory inflammatory diseaseshave already been diagnosed and have progressed to some degree. Theseresults indicate that inhibitors of a chitinase-like molecule cansuccessfully treat diseases even after chronic pathology has beenestablished.

Effects of Anti-AMCase Antibodies in Murine Asthma Models

Wild type mice were sensitized to OVA and challenged with OVA a numberof days later. One day prior to OVA challenge, mice were randomized intotwo groups and received daily doses of anti-AMCase antibodies or a serumcontrol. Similar to the symptoms of human asthma, the OVA sensitizationand challenge model results in a brisk eosinophil- and lymphocyte-richinflammatory response with prominent mucus metaplasia and goblet cellhyperplasia. Anti-AMCase antibody administration resulted in asignificant inhibition of total cell and eosinophil influx into the BALfluid of these OVA challenged lungs (FIG. 18). A similar decrease intotal and eosinophilic inflammation was noted in OVA sensitized andchallenged long tissues. Therefore, anti-AMCase inhibitedantigen-induced inflammation in an art-recognized murine model of humanasthma.

AMCase Expression in Human Lung Tissue

The observations in the art-recognized mouse model of human asthma wereextended to assessing the expression of AMCase in human lung tissue thatwas obtained at autopsy using in situ hybridization. Briefly, similarlyto the protocols described previously elsewhere herein relating to insitu hybridization for detection of mouse AMCase and Ym-1 mRNA, normaland asthmatic lung tissues were obtained through biopsies and fixed informaldehyde and processed into paraffin. Five micron sections were cut,deparaffinized, and treated with proteinase K (20 μg/ml, 37° C., 20min). Tissues were then treated with 0.1 M triethylnolamine/0.25% aceticanhydride (pH 8) for 10 min at room temperature and rinsed in PBS.

The templates for ISH probes for human AMCase were purchased fromResearch Genetics, Inc. (Huntsville, Ala.) as expressed sequence tag(EST) clones 5182357. The clone was generated using vector pCMV-SPORT6comprising T7 and SP6 promoter sequences flanking the insert fragment.The sequence of the clone matched human AMCase mRNA from nucleotide769-1538 (SEQ ID NO:14). The sequence of the clone was confirmed bynucleotide sequencing at Keck Biotechnology Laboratory at YaleUniversity. Sense and antisense RNA probes were in vitro transcribed andlabeled with a digoxigenin RNA labeling kit (Roche, Indianapolis, Ind.),denatured at 65° C., and added to commercially available hybridizationbuffer (Ambion, Austin, Tex.) at 6 ng/μl, and the hybridization mixturewas incubated with tissue overnight at 52° C. The tissues were thenwashed twice with 4×SSC for 5 minutes at room temperature, twice with2×SSC for 10 minutes at 37° C., and incubated with RNase A (10 μg/ml)for 45 minutes at 37° C. This was followed by two 10-minute washes in2×SSC at room temperature and three 20-minute washes in 0.2×SSC at 50°C. Probes were detected by overnight incubation with sheep antibodies todigoxigenin conjugated with alkaline phosphatase (Roche) followed by4-nitroblue tetrazolium chloride/5-bromo-4-chloro-3-indoylphosphate, asdescribed by the manufacturer.

Expression of AMCase in histologically normal human lung tissue(“control tissue”) was compared with AMCase expression in tissuesobtained from human patients that had succumbed due to fatal asthma(“fatal asthma”). Using in situ hybridization, AMCase mRNA was detectedin fatal asthma lung samples but not in control lung tissue using asense probe (FIGS. 19A and 19B, respectively). AMCase mRNA was localizedto epithelial cells and macrophages in fatal asthma tissue. Further, asense probe did not detect mRNA encoding AMCase in either fatal asthmaor control lung tissue, demonstrating the specificity of the probe. Inaddition, a poly-dT probe highlighted intact mRNA in both samples.

Expression of AMCase mRNA in human alveolar macrophages was alsoassessed. In situ hybridization using an AMCase antisense probedemonstrated detectable AMCase mRNA in alveolar macrophages in fatalasthma lung samples (FIG. 19E) but not in the control samples. AMCasemRNA was not detected in fatal asthma lung samples using a sense AMCaseprobe, demonstrating the specificity of the in situ hybridizationprocedure.

These data confirm that the results obtained using the art-recognizedmouse model of human asthma are in accordance with human in vivo data.That is, the data disclosed herein demonstrate, for the first time, thatAMCase mRNA expression is greatly increased, and correlated with, asthmain humans, since AMCase mRNA is present in detectable levels in fatalasthma lung tissue, but is not detectable in histologically normal lungtissue. These results further support the demonstration that expressionof chitinase-like molecules is associated with and/or mediatesinflammatory disease in mammals. Thus, the data disclosed herein furthersupport that inhibition of AMCase, using a chitinase-like moleculeinhibitor, can treat and/or prevent an inflammatory disease, such as,but not limited to, asthma, in a mammal, including, a human patient.

Discussion

The present invention is based, in part, on experimental evidencestrongly suggesting that the Th2 inflammatory responses characteristicof allergies, atopic asthma and the inflammatory and ainvay remodelingresponses associated with these disorders have evolved from the Th2inflammatory responses first developed to combat parasites and otherpathogens. Asthma and other inflammatory diseases are thus theconsequence of poorly controlled Th2 responses elicited in a parasiteand pathogen-independent manner.

A vast array of parasites and other pathogens contain chitin. It is anessential component in the exoskeletons of crustaceans and insects, thewalls of fungi, the digestive tracts of insects, the microfilarialsheath of parasitic nematodes and components of helminthic parasites.Chitin is a polymer of N-acetylglucosamine residues in a β-1,4 linkageand is the second most abundant polysaccharide in nature, behindcellulose, and has no mammalian counterpart. Chitin often comprises theexterior of parasites and other pathogens because, depending on itsthickness, it can be a rigid or flexible coating vital to protecting theorganisms from the host defenses and from the surrounding environment.Thusly, because it is present at the host/pathogen interface, chitin isa prominent antigen since it is exposed to the immune system. In fact,chitin has demonstrated to be a potent T and B cell adjuvant whichaugments immunoglobulin responses to poorly immunogenic peptides, andwhich can shift immune responses in a Th1 direction (Seferian andMartinez, 2000, Vaccine 19:661-668; Shibata et al., 2000 J. Immunol.164:1314-1321).

Given the enormous body of evidence demonstrating the co-evolution ofparasites and humans, especially in regards to host immune responses,and the complete lack of chitin in mammals, it is reasonable to believe,without intending to be bound by any particular theory, that mammalsdeveloped chitinases as an innate defense against chitin-bearingparasites. Chitinases have been identified in mammals. Chitotriosidaseand YKL-39 have been described in humans, YM protein has been describedin the mouse, and acidic mammalian chitinase (AMCase), oviductin, andYKL-40 have been described in both mice and humans (Boot et al., 2001,J. Biol. Chem. 276:6770-6778; Boot et al., 1998, J. Biol. Chem.273:25680-25685; Ward et al., 2001, Am. J. Pathol. 158:323-332; Chang etal., 2001, J. Biol. Chem. 276:17497-17506; Jin et al., 1998, Genetics54:316-322; Bleau et al., 1999, EXS 87:211-221). Despite conflictingreports and some sequence homology to microbial chitinases, onlychitotriosidase and AMCase have demonstrated true chitinase activity(Boot et al., 2001, J. Biol. Chem. 276:6770-6778). Of the mammalianchitinases, both YM-1 and AMCase have been identified as havingchemotactic and growth factor-like properties. YM-1 is a single chainpeptide of 45 kDa that readily forms crystals under physiologicalconditions. YM family proteins have been identified as both eosinophilicand a CD4⁺ T-cell attractant, as well as having lectin activity (Owhashiet al., 2000, J. Biol. Chem. 275:12791286; Chang et al., 2001, J. Biol.Chem. 276:17497-17506). AMCase is a 50 kDa protein that demonstrateschitinase activity. It has also been reported as a fibroblast growthpromoting agent (Guoping et al., 1997, J. Cell. Biochem. 67:257-264). Asdemonstrated by the data disclosed herein, both YM family proteins andAMCase are highly expressed in the lung of IL-13 induced, and Th2mediated asthma models.

Without wishing to be bound by any particular theory, when viewed incombination with the role of chitin in the asthmatic condition, thenovel discovery of the role of chitinases in asthma and otherrespiratory inflammatory diseases may be viewed as follows. In anon-atopic, non-asthmatic subject, exposure to chitin as an antigen,possibly after exposure to a parasite or fungus, polarizes the immuneresponse towards a Th1 pathway, which may or may not effectively combatthe parasite. However, in the atopic subject or one genetically disposedto asthma or atopy, IL-13 and/or IL-4 are produced which upregulate theexpression of YM, AMCase, and possibly other chitinases andchitinase-like molecules. Chitin, as detailed earlier, augments IgEproduction. YM family proteins, alone or in combination with othermolecules, possess eosinophil and CD4⁺ T cell chemotactic properties.Therefore, YM proteins can attract eosinophils to the lung, as well ascause tissue damage mediated by its carbohydrate-binding andcrystal-forming characteristics. AMCase eventually degrades the chitinto eliminate the pathogen. Because of the Th1 inducing properties ofchitin, AMCase activity and subsequent chitin degradation steers theimmune system towards a Th2 response characteristic of atopy andasthmatic inflammatory diseases.

The present invention includes methods and therapeutics for thetreatment of asthma, COPD, and other inflammatory diseases usingchitinase-like molecule inhibitors. The invention further comprisesmethods of identifying novel therapeutics for the treatment of asthma,COPD, and other inflammatory diseases relating to IL-13, IL-4 chitinasesand chitinase-like molecule inhibitors.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety.

While this invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention. The appendedclaims are intended to be construed to include all such embodiments andequivalent variations.

1-41. (canceled)
 42. A method of treating an inflammatory disease in amammal, wherein said disease is associated with an increased level of achitinase-like molecule, comprising administering an effective amount ofa chemical compound inhibitor of the chitinase-like molecule to saidmammal, thereby treating said inflammatory disease.
 43. The method ofclaim 42, wherein said mammal is a human.
 44. The method of claim 42,wherein said chitinase-like molecule is selected from the groupconsisting of a YM-1, a YM-2, an acidic mammalian chitinase (AMCase), anoviductal glycoprotein 1, a cartilage glycoprotein 1, a chitotriosidase,a mucin 9, a cartilage glycoprotein-39, YKL-40, chitinase 3-like-1 and achondrocyte protein
 39. 45. The method of claim 42, wherein saidchemical compound is selected from the group consisting of allosamidin,glucoallosamidin A, glucoallosamidin B, methyl-N-demethylallosamidin,demethylallosamidin, didemthylallosamidin, stylogaunidine, astyloguanidine derivative, dipeptide cyclo-(L-Arg-D-Pro), dipeptidecyclo-(L-Arg-L-Pro), dipeptide cyclo-(D-Arg-D-Pro), dipeptidecyclo-(D-Arg-L-Pro), riboflavin, a flavin derivative, copper, zinc, andmercury.
 46. The method of claim 42, wherein said inflammatory diseaseis selected from the group consisting of asthma, chronic obstructivepulmonary disease, interstitial lung disease, chronic obstructive lungdisease, chronic bronchitis, eosinophilic bronchitis, eosinophilicpneumonia, pneumonia, inflammatory bowel disease, atopic dermatitis,atopy, allergy, allergic rhinitis, idiopathic pulmonary fibrosis,scleroderma, and emphysema.
 47. A method of preventing an inflammatorydisease in a mammal, wherein said disease is associated with anincreased level of a chitinase-like molecule, comprising administeringan effective amount of a chemical compound inhibitor of thechitinase-like molecule to said mammal, thereby preventing saidinflammatory disease.
 48. The method of claim 47, wherein said mammal isa human.
 49. The method of claim 47, wherein said chitinase-likemolecule is selected from the group consisting of a YM-1, a YM-2, anAMCase, an oviductal glycoprotein 1, a cartilage glycoprotein 1, achitotriosidase, a mucin 9, a cartilage glycoprotein-39, YKL-40,chitinase 3-like-1 and a chondrocyte protein
 39. 50. The method of claim47, wherein said inflammatory disease is selected from the groupconsisting of asthma, chronic obstructive pulmonary disease,interstitial lung disease, chronic obstructive lung disease, chronicbronchitis, eosinophilic bronchitis, eosinophilic pneumonia, pneumonia,inflammatory bowel disease, atopic dermatitis, atopy, allergy, allergicrhinitis, idiopathic pulmonary fibrosis, scleroderma, and emphysema. 51.The method of claim 47, wherein said chemical compound is selected fromthe group consisting of allosamidin, glucoallosamidin A,glucoallosamidin B, methyl-N-demethylallosamidin, demethylallosamidin,didemthylallosamidin, styloguanidine, a styloguanidine derivative,dipeptide cyclo-(L-Arg-D-Pro), dipeptide cyclo-(L-Arg-L-Pro), dipeptidecyclo-(D-Arg-D-Pro), dipeptide cyclo-(D-Arg-L-Pro), riboflavin, a flavinderivative, copper, zinc, and mercury.
 52. A method of treating aninflammatory disease in a mammal, wherein said disease is associatedwith an increased level of chitinase, comprising administering aneffective amount of a chemical compound inhibitor of chitinase to saidmammal, thereby treating said inflammatory disease.
 53. The method ofclaim 52, wherein said mammal is a human.
 54. The method of claim 52,wherein said chitinase is AMCase or chitotriosidase.
 55. The method ofclaim 52, wherein said chemical compound is selected from the groupconsisting of allosamidin, glucoallosamidin A, glucoallosamidin B,methyl-N-demethylallosamidin, demethylallosamidin, didemthylallosamidin,styloguanidine, a styloguanidine derivative, dipeptidecyclo-(L-Arg-D-Pro), dipeptide cyclo-(L-Arg-L-Pro), dipeptidecyclo-(D-Arg-D-Pro), dipeptide cyclo-(D-Arg-L-Pro), riboflavin, a flavinderivative, copper, zinc, and mercury.
 56. The method of claim 52,wherein said inflammatory disease is selected from the group consistingof asthma, chronic obstructive pulmonary disease, interstitial lungdisease, chronic obstructive lung disease, chronic bronchitis,eosinophilic bronchitis, eosinophilic pneumonia, pneumonia, inflammatorybowel disease, atopic dermatitis, atopy, allergy, allergic rhinitis,idiopathic pulmonary fibrosis, scleroderma, and emphysema.
 57. A methodfor treating an inflammatory disease in a mammal, wherein said diseaseis associated with an increased level of interleukin-13, comprisingadministering an effective amount of chemical compound inhibitor of achitinase-like molecule to said mammal, thereby treating saidinflammatory disease.
 58. The method of claim 57, wherein said mammal isa human.
 59. The method of claim 57, wherein said chitinase-likemolecule is selected from the group consisting of a YM-1, a YM-2, anAMCase, an oviductal glycoprotein 1, a cartilage glycoprotein 1, achitotriosidase, a mucin 9, a cartilage glycoprotein-39, YKL-40,chitinase 3-like-1 and a chondrocyte protein
 39. 60. The method of claim57, wherein said inflammatory disease is selected from the groupconsisting of asthma and chronic obstructive pulmonary disease.
 61. Themethod of claim 57, wherein said chemical compound is selected from thegroup consisting of allosamidin, glucoallosamidin A, glucoallosamidin B,methyl-N-demethylallosamidin, demethylallosamidin, didemthylallosamidin,styloguanidine, a styloguanidine derivative, dipeptidecyclo-(L-Arg-D-Pro), dipeptide cyclo-(L-Arg-L-Pro), dipeptidecyclo-(D-Arg-D-Pro), dipeptide cyclo-(D-Arg-L-Pro), riboflavin, a flavinderivative, copper, zinc, and mercury.
 62. A method for treating aninflammatory disease in a mammal, wherein said disease is associatedwith a Th2 inflammatory response, comprising administering an effectiveamount of a chemical compound inhibitor of a chitinase-like molecule tosaid mammal, thereby treating said inflammatory disease.
 63. The methodof claim 62, wherein said mammal is a human.
 64. The method of claim 62,wherein said chitinase-like molecule is selected from the groupconsisting of a YM-1, a YM-2, an AMCase, an oviductal glycoprotein 1, acartilage glycoprotein 1, a chitotriosidase, a mucin 9, a cartilageglycoprotein-39, YKL-40, chitinase 3-like-I and a chondrocyte protein39.
 65. The method of claim 62, wherein inflammatory said disease isselected from the group consisting of asthma, chronic obstructivepulmonary disease, interstitial lung disease, chronic obstructive lungdisease, chronic bronchitis, eosinophilic bronchitis, eosinophilicpneumonia, pneumonia, inflammatory bowel disease, atopic dermatitis,atopy, allergy, allergic rhinitis, idiopathic pulmonary fibrosis,scleroderma, and emphysema.
 66. The method of claim 62, wherein saidchemical compound is selected from the group consisting of allosamidin,glucoallosamidin A, glucoallosamidin B, methyl-N-demethylallosamidin,demethylallosamidin, didemthylallosamidin, styloguanidine, astyloguanidine derivative, dipeptide cyclo-(L-Arg-D-Pro), dipeptidecyclo-(L-Arg-L-Pro), dipeptide cyclo-(D-Arg-D-Pro), dipeptidecyclo-(D-Arg-L-Pro), riboflavin, a flavin derivative, copper, zinc, andmercury.
 67. A method of inhibiting an activity of a chitinase-likemolecule in a mammal, comprising administering an effective amount of achemical compound inhibitor of the chitinase-like molecule to saidmammal.
 68. The method of claim 67, wherein said mammal is sufferingfrom an inflammatory disease associated with an increased level of achitinase-like molecule.
 69. The method of claim 68, wherein saidinflammatory disease is selected from the group consisting of asthma,chronic obstructive pulmonary disease, interstitial lung disease,chronic obstructive lung disease, chronic bronchitis, eosinophilicbronchitis, eosinophilic pneumonia, pneumonia, inflammatory boweldisease, atopic dermatitis, atopy, allergy, allergic rhinitis,idiopathic pulmonary fibrosis, scleroderma, and emphysema.
 70. Themethod of claim 67, wherein said mammal is a human.
 71. The method ofclaim 67, wherein said chitinase-like molecule is selected from thegroup consisting of a YM-1, a YM-2, an acidic mammalian chitinase(AMCase), an oviductal glycoprotein 1, a cartilage glycoprotein 1, achitotriosidase, a mucin 9, a cartilage glycoprotein-39, YKL-40,chitinase 3-like-1 and a chondrocyte protein
 39. 72. The method of claim67, wherein said chemical compound is selected from the group consistingof allosamidin, glucoallosamidin A, glucoallosamidin B,methyl-N-demethylallosamidin, demethylallosamidin, didemthylallosamidin,styloguanidine, a styloguanidine derivative, dipeptidecyclo-(L-Arg-D-Pro), dipeptide cyclo-(L-Arg-L-Pro), dipeptidecyclo-(D-Arg-D-Pro), dipeptide cyclo-(D-Arg-L-Pro), riboflavin, a flavinderivative, copper, zinc, and mercury.